US20070031843A1

US20070031843A1 - Bioinformatically detectable group of novel regulatory bacterial and bacterial associated oligonucleotides and uses thereof

Info

Publication number: US20070031843A1
Application number: US10/709,691
Authority: US
Inventors: Itzhak Bentwich; Amir Avniel
Original assignee: Rosetta Genomics Ltd
Current assignee: Rosetta Genomics Ltd
Priority date: 2004-04-02
Filing date: 2004-05-24
Publication date: 2007-02-08
Also published as: US7943754B2

Abstract

The present invention relates to a first group of novel bacterial and human associated oligonucleotides, here identified as “Genomic Address Messenger” or “GAM” oligonucleotide, and a second group of novel operon-like bacterial and human polynucleotides, here identified as “Genomic Record” or “GR” polynucleotide. GAM oligonucleotides selectively inhibit translation of known “target” genes, many of which are known to be involved in various bacterial infections. Nucleic acid molecules are provided respectively encoding 21,916 bacterial and 6,100 human GAM precursor oligonucleotides, and 6,056 bacterial and 430 human GR polynucleotides, as are vectors and probes both comprising the nucleic acid molecules, and methods and systems for detecting GAM oligonucleotides and GR polynucleotides and specific functions and utilities thereof, for detecting expression of GAM oligonucleotides and GR polynucleotides, and for selectively enhancing and selectively inhibiting translation of the respective target genes thereof.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. patent Application Ser. No. 10/708,951, filed 2 Apr. 2004, entitled “Bioinformatically Detectable Group of Novel Regulatory Bacterial and Bacterial Associated Oligonucleotides and Uses Thereof ”, the disclosure of which is hereby incorporated by reference and claims priority therefrom; This application also is a continuation in part of U.S. Provisional Patent Application Ser. No. 60/521,433 filed 26 Apr. 2004, entitled “A Microarray for the Detection of MicroRNA Oligonucleotides”, the disclosure of which is hereby incorporated by reference and claims priority therefrom.

REFERENCES CITED

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Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188-200.
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Hamosh A, Scott A F, Amberger J, Bocchini C, Valle D and McKusick V A. (2002). Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Res. 30: 52-55.
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Krichevsky, A. M., King, K. S., Donahue, C. P., Khrapko, K., and Kosik, K. S. (2003). A microRNA array reveals extensive regulation of microRNAs during brain development. RNA. 9, 1274-1281.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853-858.
Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858-862.
Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E. L. (1996). Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14, 1675-1680.
Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911-940.
Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R., and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906.
Southern, E. M. (1992). Detection of specific sequences among DNA fragments separated by gel electrophoresis. 1975. Biotechnology 24, 122-139.
Tom M. Mitchell, Machine Learning, McGraw Hill, 1997.
Wightman, B., Ha, I., and Ruvkun, G. (1993). Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855-862.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to a group of bioinformatically detectable novel bacterial oligonucleotides and to a group of bioinformatically detectable novel human oligonucleotides associated with bacterial infections, both are identified here as “Genomic Address Messenger” (GAM) oligonucleotides.
All of abovementioned oligonucleotides are believed to be related to the microRNA (miRNA) group of oligonucleotides.
2. Description of Prior Art
miRNA oligonucleotides are short ˜22 nucleotide (nt)-long, non-coding, regulatory RNA oligonucleotides that are found in a wide range of species. miRNA oligonucleotides are believed to function as specific gene translation repressors and are sometimes involved in cell differentiation.
The ability to detect novel miRNA oligonucleotides is limited by the methodologies used to detect such oligonucleotides. All miRNA oligonucleotides identified so far either present a visibly discernable whole body phenotype, as do Lin-4 and Let-7 (Wightman, B., Ha, I., and Ruvkun, G., Cell 75: 855-862 (1993); Reinhart et al. Nature 403: 901-906 (2000)), or produce sufficient quantities of RNA so as to be detected by standard molecular biological techniques.
Ninety-three miRNA oligonucleotides have been discovered in several species (Lau et al., Science 294: 858-862 (2001), Lagos-Quintana et al., Science 294: 853-858 (2001)) by sequencing a limited number of clones (300 by Lau and 100 by Lagos-Quintana) of size-fractionated small segments of RNA. miRNAs that were detected in these studies therefore represent the more prevalent among the miRNA oligonucleotide family and cannot be much rarer than 1% of all small ˜20 nt-long RNA oligonucleotides.
The aforementioned studies provide no basis for the detection of miRNA oligonucleotides which either do not present a visually discernable whole body phenotype, or are rare (e.g. rarer than 0.1% of all of the size-fractionated, ˜20 nt-long RNA segments that were expressed in the tissues examined), and therefore do not produce large enough quantities of RNA to be detected by standard biological techniques.
To date, miRNA oligonucleotides have not been detected in bacteria.
The following U.S. Patents relate to bioinformatic detection of genes: U.S. Pat. No. 348,935, entitled “Statistical algorithms for folding and target accessibility prediction and design of nucleic acids”, U.S. Pat. No. 6,369,195, entitled “Prostate-specific gene for diagnosis, prognosis and management of prostate cancer”, and U.S. Pat. No. 6,291,666 entitled “Spike tissue-specific promoter”, each of which is hereby incorporated by reference herein.
Brief Description of Sequence Listing, Tables and Computer Program Listing
A sequence listing is attached to the present invention, comprising 4,254,670 genomic sequences, is contained in a file named SEQ_LIST.txt (720288 KB, 18 May 2004), and is hereby incorporated by reference herein.
Tables relating to genomic sequences are attached to the present application, appear in the following files (size, creation date) included on CD, incorporated herein: TABLE_—1.txt (28.3 MB, 18 May 2004), TABLE_—2.txt (350 MB, 18 May 2004), TABLE_—3.txt (5.64 MB, 18 May 2004), TABLE_—4.txt (17.1 MB, 18 May 2004), TABLE_—5.txt (5.04 MB, 18 May 2004), TABLE_—6.txt (536 MB, 18 May 2004), TABLE_—7_A.txt (619 MB, 18 May 2004), TABLE_—7_B.txt (340 MB, 18 May 2004), TABLE_—8_A.txt (619 MB, 18 May 2004), TABLE_—8_B.txt (619 MB, 18 May 2004), TABLE_—8_C.txt (619 MB, 18 May 2004), TABLE_—8_D.txt (457 MB, 18 May 2004), TABLE_—9.txt (654 MB, 18 May 2004), TABLE_—10.txt (49.1 MB, 18 May 2004), and TABLE_—11.txt (79.8 MB, 18 May 2004), all of which are incorporated by reference herein. Further, additional tables relating to genomic sequences are attached to the present application, appear in the following files (size, creation date) attached to the application, incorporated herein: TABLE_—12.txt (41.1 KB, 18 May 2004) and TABLE_—13.txt (46.9 KB, 18 May 2004), are incorporated by reference herein.
A computer program listing constructed and operative in accordance with a preferred embodiment of the present invention is enclosed on an electronic medium in computer readable form, and is hereby incorporated by reference herein. The computer program listing is contained in 7 files, the name, sizes and creation date of which are as follows: AUXILARY_FILES.txt (117K, 14 Nov. 2003); EDIT_DISTANCE.txt (144K, 24 Nov. 2003); FIRST-K.txt (96K, 24 Nov. 2003); HAIRPIN_PREDICTION.txt (19K, 25 Mar. 2004); TWO_PHASED_SIDE_SELECTOR.txt (4K, 14 Nov. 2003); TWO_PHASED_PREDICTOR.txt (74K, 14 Nov. 2003), and BS_CODE.txt (118K, 11 May 2004).

SUMMARY OF THE INVENTION

The present invention relates to a novel group of 3,873 bioinformatically detectable bacterial regulatory RNA oligonucleotides, which repress expression of human target genes, by means of complementary hybridization to binding sites in untranslated regions of these target genes. It is believed that this novel group of bacterial oligonucleotides represents a pervasive bacterial mechanism of attacking a host, and therefore knowledge of this novel group of bacterial oligonucleotides may be useful in preventing and treating bacterial diseases.
Additionally, the present invention relates to a novel group of 4,363 bioinformatically detectable human regulatory RNA oligonucleotides, which repress expression of human target genes associated with the bacterial infection, by means of complementary hybridization to binding sites in untranslated regions of these target genes. It is believed that this novel group of human oligonucleotides represents a pervasive novel host response mechanism, and therefore knowledge of this novel group of human oligonucleotides may be useful in preventing and treating bacterial diseases.
Furthermore, the present invention relates to a novel group of 24,160 bioinformatically detectable bacterial regulatory RNA oligonucleotides, which repress expression of bacterial target genes, by means of complementary hybridization to binding sites in untranslated regions of these bacterial target genes. It is believed that this novel group of bacterial oligonucleotides represents a pervasive novel internal bacterial regulation mechanism, and therefore knowledge of this novel group of bacterial oligonucleotides may be useful in preventing and treating bacterial diseases.
In addition, the present invention relates to a novel group of 6,100 bioinformatically detectable human regulatory RNA oligonucleotides, which repress expression of bacterial target genes, by means of complementary hybridization to binding sites in untranslated regions of these bacterial target genes. It is believed that this novel group of human oligonucleotides represents a pervasive novel antibacterial host defense mechanism, and therefore knowledge of this novel group of human oligonucleotides may be useful in preventing and treating bacterial diseases.
Also disclosed are 6,056 novel microRNA-cluster like bacterial polynucleotides and 430 novel microRNA-cluster like human polynucleotides, both referred to here as Genomic Record (GR) polynucleotides.
In various preferred embodiments, the present invention seeks to provide improved method and system for detection and prevention of bacterial diseases, which are mediated by this group of novel oligonucleotides.
Accordingly, the invention provides several substantially pure nucleic acids (e.g., genomic DNA, cDNA or synthetic DNA) each comprising a novel GAM oligonucleotide, vectors comprising the DNAs, probes comprising the DNAs, a method and system for selectively modulating translation of known target genes utilizing the vectors, and a method and system utilizing the GAM probes to modulate expression of GAM target genes.
The present invention represents a scientific breakthrough, disclosing novel miRNA-like oligonucleotides the number of which is dramatically larger than previously believed existed. Prior-art studies reporting miRNA oligonucleotides ((Lau et al., Science 294:858-862 (2001), Lagos-Quintana et al., Science 294: 853-858 (2001)) discovered 93 miRNA oligonucleotides in several species, including 21 in human, using conventional molecular biology methods, such as cloning and sequencing.
Molecular biology methodologies employed by these studies are limited in their ability to detect rare miRNA oligonucleotides, since these studies relied on sequencing of a limited number of clones (300 clones by Lau and 100 clones by Lagos-Quintana) of small segments (i.e. size-fractionated) of RNA. miRNA oligonucleotides detected in these studies therefore, represent the more prevalent among the miRNA oligonucleotide family, and are typically not be much rarer than 1% of all small ˜20 nt-long RNA oligonucleotides present in the tissue from the RNA was extracted.
Recent studies state the number of miRNA oligonucleotides to be limited, and describe the limited sensitivity of available methods for detection of miRNA oligonucleotides: “The estimate of 255 human miRNA oligonucleotides is an upper bound implying that no more than 40 miRNA oligonucleotides remain to be identified in mammals” (Lim et al., Science, 299:1540 (2003)); “Estimates place the total number of vertebrate miRNA genes at about 200-250” (Ambros et al. Curr. Biol. 13:807-818 (2003)); and “Confirmation of very low abundance miRNAs awaits the application of detection methods more sensitive than Northern blots” (Ambros et al. Curr. Biol. 13:807-818 (2003)).
The oligonucleotides of the present invention represent a revolutionary new dimension of genomics and of biology: a dimension comprising a huge number of non-protein-coding oligonucleotides which modulate expression of thousands of proteins and are associated with numerous major diseases. This new dimension disclosed by the present invention dismantles a central dogma that has dominated life-sciences during the past 50 years, a dogma which has emphasized the importance of protein-coding regions of the genome, holding non-protein-coding regions to be of little consequence, often dubbing them “junk DNA”.
Indeed, only in November, 2003 has this long held belief as to the low importance of non-protein-coding regions been vocally challenged. As an example, an article titled “The Unseen Genome-Gems in the Junk” (Gibbs, W. W. Sci. Am. 289:46-53 (2003)) asserts that the failure to recognize the importance of non-protein-coding regions “may well go down as one of the biggest mistakes in the history of molecular biology.” Gibbs further asserts that “what was damned as junk because it was not understood, may in fact turn out to be the very basis of human complexity.” The present invention provides a dramatic leap in understanding specific important roles of non-protein-coding regions.
An additional scientific breakthrough of the present invention is a novel conceptual model disclosed by the present invention, which conceptual model is preferably used to encode in a genome the determination of cell differentiation, utilizing oligonucleotides and polynucleotides of the present invention.
Using the bioinformatic engine of the present invention, 21,916 bacterial GAM oligonucleotides and their respective precursors and targets have been detected and 6,100 human GAM oligonucleotides and their respective precursors and targets have been detected. These bioinformatic predictions are supported by robust biological studies. Microarray experiments validated expression of 346 of the human GAM oligonucleotides of the present invention. Of these, 311 received an extremely high score: over six standard deviations higher than the background “noise” of the microarray, and over two standard deviations above their individual “mismatch” control probes and 33 received a high score: over four standard deviations higher than the background “noise” of the microarray. Further, 38 GAM oligonucleotides were sequenced.
In various preferred embodiments, the present invention seeks to provide an improved method and system for specific modulation of the expression of specific target genes involved in significant human diseases. It also provides an improved method and system for detection of the expression of novel oligonucleotides of the present invention, which modulate these target genes. In many cases, the target genes may be known and fully characterized, however in alternative embodiments of the present invention, unknown or less well characterized genes may be targeted.
A “Nucleic acid” is defined as a ribonucleic acid (RNA) molecule, or a deoxyribonucleic acid (DNA) molecule, or complementary deoxyribonucleic acid (cDNA), comprising either naturally occurring nucleotides or non-naturally occurring nucleotides.
“Substantially pure nucleic acid”, “Isolated Nucleic Acid”, “Isolated Oligoucleotide” and “Isolated Polynucleotide” are defined as a nucleic acid that is free of the genome of the organism from which the nucleic acid is derived, and include, for example, a recombinant nucleic acid which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic nucleic acid of a prokaryote or eukaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other nucleic acids.
An “Oligonucleotide” is defined as a nucleic acid comprising 2-139 nts, or preferably 16-120 nts. A “Polynucleotide” is defined as a nucleic acid comprising 140-5000 nts, or preferably 140-1000 nts.
A “Complementary” sequence is defined as a first nucleotide sequence which reverses complementary of a second nucleotide sequence: the first nucleotide sequence is reversed relative to a second nucleotide sequence, and wherein each nucleotide in the first nucleotide sequence is complementary to a corresponding nucleotide in the second nucleotide sequence (e.g. ATGGC is the complementary sequence of GCCAT).
“Hybridization”, “Binding” and “Annealing” are defined as hybridization, under in vivo physiological conditions, of a first nucleic acid to a second nucleic acid, which second nucleic acid is at least partially complementary to the first nucleic acid.
A “Hairpin Structure” is defined as an oligonucleotide having a nucleotide sequence that is 50-140 nts in length, the first half of which nucleotide sequence is at least partially complementary to the second part thereof, thereby causing the nucleic acid to fold onto itself, forming a secondary hairpin structure.
A “Hairpin-Shaped Precursor” is defined as a Hairpin Structure which is processed by a Dicer enzyme complex, yielding an oligonucleotide which is about 19 to about 24 nts in length.
“Inhibiting translation” is defined as the ability to prevent synthesis of a specific protein encoded by a respective gene by means of inhibiting the translation of the mRNA of this gene. For example, inhibiting translation may include the following steps: (1) a DNA segment encodes an RNA, the first half of whose sequence is partially complementary to the second half thereof; (2) the precursor folds onto itself forming a hairpin-shaped precursor; (3) a Dicer enzyme complex cuts the hairpin-shaped precursor yielding an oligonucleotide that is approximately 22 nt in length; (4) the oligonucleotide binds complementarily to at least one binding site, having a nucleotide sequence that is at least partially complementary to the oligonucleotide, which binding site is located in the mRNA of a target gene, preferably in the untranslated region (UTR) of a target gene, such that the binding inhibits translation of the target protein.
A “Translation inhibitor site” is defined as the minimal nucleotide sequence sufficient to inhibit translation.
The present invention describes novel GAM oligonucleotides, detected using a bioinformatic engine described hereinabove. The ability of this detection engine has been demonstrated using stringent algorithmic criteria, showing that the engine has both high sensitivity, indicated by the high detection rate of published miRNA oligonucleotides and their targets, as well as high specificity, indicated by the low amount of “background” hairpin candidates passing its filters. Laboratory tests, based both on sequencing of predicted GAM oligonucleotides and on microarray experiments, validated 381 of the GAM oligonucleotides in the present invention. Further, almost all of the bacterial target genes (6,141 of the 7,351) and almost all of the human target genes (64 out of 76) described in the present invention are bound by one or more of the 381 human GAM oligonucleotides validated by the microarray experiments.
There is thus provided in accordance with a preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable first oligonucleotide which is a portion of a mRNA transcript of a target gene, and anneals to a second oligonucleotide that is endogenously processed from a hairpin precursor, wherein binding of the first oligonucleotide to the second oligonucleotide represses expression of the target gene, and wherein nucleotide sequence of the second nucleotide is selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2337129-4223628.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Bordetella pertussis infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 2.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Brucella suis 1330 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 3.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydia trachomatis infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 4.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae AR39 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 5.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae CWL029 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 6.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae J138 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 7.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae TW-183 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 8.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Coxiella burnetii RSA 493 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 9.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Escherichia coli CFT073 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 10.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Haemophilus influenzae Rd infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 11.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Leptospira interrogans serovar lai str. 56601 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 12.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Listeria monocytogenes EGD-e infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 13.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Mycobacterium avium subsp. paratuberculosis infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 14.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Mycobacterium bovis subsp bovis AF2122/97 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 15.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Mycobacterium leprae infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 16.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Mycobacterium tuberculosis CDC1551 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 17.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Mycobacterium tuberculosis H37Rv infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 18.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Neisseria meningitidis MC58 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 19.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Neisseria meningitidis Z2491 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 20.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Pseudomonas aeruginosa PA01 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 21.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Pseudomonas putida KT2440 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 22.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Rickettsia prowazekii infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 23.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Salmonella enterica enterica serovar Typhi infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 24.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Salmonella enterica enterica serovar Typhi Ty2 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 25.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Salmonella typhimurium LT2 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 26.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Shigella flexneri 2a str. 2457T infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 27.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Shigella flexneri 2a str. 301 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 28.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Staphylococcus aureus subsp. aureus Mu50 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 29.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Staphylococcus aureus subsp. aureus MW2 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 30.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Staphylococcus aureus subsp. aureus N315 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 31.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pneumoniae R6 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 32.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pneumoniae TIGR4 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 33.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pyogenes M1 GAS infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 34.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pyogenes MGAS315 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 35.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pyogenes MGAS8232 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 36.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Streptococcus pyogenes SSI-1 infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 37.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Treponema pallidum subsp. pallidum str. Nichols infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 38.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Yersinia pestis infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 39.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Yersinia pestis KIM infection, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 40.
There is additionally provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving a tissue in which a protein is pathologically expressed to an undesirable extent, the protein having a messenger RNA, the method including: providing a material which modulates activity of a microRNA oligonucleotide which binds complementarily to a segment of the messenger RNA, and introducing the material into the tissue, causing modulation of the activity of the microRNA oligonucleotide and thereby modulating expression of the protein in a desired manner.
There is moreover provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving tissue in which a protein is pathologically expressed to an undesirable extent, the protein having a messenger RNA, the method including: providing a material which at least partially binds a segment of the messenger RNA that is bound complementarily by a microRNA oligonucleotide, thereby modulating expression of the protein, and introducing the material into the tissue, thereby modulating expression of the protein.
There is further provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving a tissue in which a protein is pathologically over-expressed, the protein having a messenger RNA, the method including: providing a microRNA oligonucleotide which binds complementarily to a segment of the messenger RNA, and introducing the microRNA oligonucleotide into the tissue, causing the microRNA oligonucleotide to bind complementarily to a segment of the messenger RNA and thereby inhibit expression of the protein.
There is still further provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving a tissue in which a protein is pathologically over-expressed, the protein having a messenger RNA, the method including: providing a chemically-modified microRNA oligonucleotide which binds complementarily to a segment of the messenger RNA, and introducing the chemically-modified microRNA oligonucleotide into the tissue, causing the microRNA oligonucleotide to bind complementarily to a segment of the messenger RNA and thereby inhibit expression of the protein.
There is additionally provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving a tissue in which a protein is pathologically under-expressed, the protein having a messenger RNA, the method including: providing an oligonucleotide that inhibits activity of a microRNA oligonucleotide which binds complementarily to a segment of the messenger RNA, and introducing the oligonucleotide into the tissue, causing inhibition of the activity of the microRNA oligonucleotide and thereby promotion of translation of the protein.
There is moreover provided in accordance with another preferred embodiment of the present invention a method for treatment of a disease involving a tissue in which a protein is pathologically under-expressed, the protein having a messenger RNA, the method including: providing a chemically-modified oligonucleotide that inhibits activity of a microRNA oligonucleotide which binds complementarily to a segment of the messenger RNA, and introducing the chemically-modified oligonucleotide into the tissue, causing inhibition of the activity of the microRNA oligonucleotide and thereby promotion of translation of the protein.
There is further provided in accordance with another preferred embodiment of the present invention a method for diagnosis of a disease involving a tissue in which a protein is expressed to abnormal extent, the protein having a messenger RNA, the method including: assaying a microRNA oligonucleotide which at least partially binds a segment of the messenger RNA and modulates expression of the protein, thereby providing an indication of at least one parameter of the disease.
There is still further provided in accordance with another preferred embodiment of the present invention a method for detection of expression of an oligonucleotide, the method including: determining a first nucleotide sequence of a first oligonucleotide, which first nucleotide sequence is not complementary to a genome of an organism, receiving a second nucleotide sequence of a second oligonucleotide whose expression is sought to be detected, designing a third nucleotide sequence that is complementary to the second nucleotide sequence of the second oligonucleotide, and a fourth nucleotide sequence that is complementary to a fifth nucleotide sequence which is different from the second nucleotide sequence of the second oligonucleotide by at least one nucleotide, synthesizing a first oligonucleotide probe having a sixth nucleotide sequence including the third nucleotide sequence followed by the first nucleotide sequence of the first oligonucleotide, and a second oligonucleotide probe having a seventh nucleotide sequence including the fourth nucleotide sequence followed by the first nucleotide sequence of the first oligonucleotide, locating the first oligonucleotide probe and the second oligonucleotide probe on a microarray platform, receiving an RNA test sample from at least one tissue of the organism, obtaining size-fractionated RNA from the RNA test sample, amplifying the size-fractionated RNA, hybridizing the adaptor-linked RNA with the first and second oligonucleotide probes on the microarray platform, and determining expression of the first oligonucleotide in the at least one tissue of the organism, based at least in part on the hybridizing.
There is additionally provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated polynucleotide which is endogenously processed into a plurality of hairpin-shaped precursor oligonucleotides, each of which is endogenously processed into a respective oligonucleotide, which in turn anneals to a portion of a mRNA transcript of a target gene, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene.
There is moreover provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the target gene does not encode a protein.
There is further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein a function of the oligonucleotide includes modulation of cell type.
There is still further provided in accordance with another preferred embodiment of the present invention a bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of the oligonucleotide to the mRNA transcript represses expression of the target gene, and wherein the oligonucleotide is maternally transferred by a cell to at least one daughter cell of the cell, and a function of the oligonucleotide includes modulation of cell type of the daughter cell.
There is additionally provided in accordance with another preferred embodiment of the present invention a method for bioinformatic detection of microRNA oligonucleotides, the method including: bioinformatically detecting a hairpin-shaped precursor oligonucleotide, bioinformatically detecting an oligonucleotide which is endogenously processed from the hairpin-shaped precursor oligonucleotide, and bioinformatically detecting a target gene of the oligonucleotide wherein the oligonucleotide anneals to at least one portion of a mRNA transcript of the target gene, and wherein the binding represses expression of the target gene, and the target gene is associated with a disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a simplified diagram illustrating a mode by which an oligonucleotide of a novel group of oligonucleotides of the present invention modulates expression of known target genes;
FIG. 2 is a simplified block diagram illustrating a bioinformatic oligonucleotide detection system capable of detecting oligonucleotides of the novel group of oligonucleotides of the present invention, which system is constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 3 is a simplified flowchart illustrating operation of a mechanism for training of a computer system to recognize the novel oligonucleotides of the present invention, which mechanism is constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 4A is a simplified block diagram of a non-coding genomic sequence detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 4B is a simplified flowchart illustrating operation of a non-coding genomic sequence detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 5A is a simplified block diagram of a hairpin detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 5B is a simplified flowchart illustrating operation of a hairpin detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 6A is a simplified block diagram of a Dicer-cut location detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 6B is a simplified flowchart illustrating training of a Dicer-cut location detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 6C is a simplified flowchart illustrating operation of a Dicer-cut location detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 7A is a simplified block diagram of a target gene binding site detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 7B is a simplified flowchart illustrating operation of a target gene binding site detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 8 is a simplified flowchart illustrating operation of a function and utility analyzer constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 9 is a simplified diagram describing a novel bioinformatically-detected group of regulatory polynucleotides, referred to here as Genomic Record (GR) polynucleotides, each of which encodes an “operon-like” cluster of novel microRNA-like oligonucleotides, which in turn modulate expression of one or more target genes;
FIG. 10 is a block diagram illustrating different utilities of novel oligonucleotides and novel operon-like polynucleotides, both of the present invention;
FIGS. 11A and 11B are simplified diagrams which, when taken together, illustrate a mode of oligonucleotide therapy applicable to novel oligonucleotides of the present invention;
FIG. 12A is a bar graph illustrating performance results of a hairpin detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 12B is a line graph illustrating accuracy of a Dicer-cut location detector constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 12C is a bar graph illustrating performance results of the target gene binding site detector 118, constructed and operative in accordance with a preferred embodiment of the present invention.
FIG. 13 is a summary table of laboratory results validating expression of novel human oligonucleotides detected by a bioinformatic oligonucleotide detection engine constructed and operative in accordance with a preferred embodiment of the present invention, thereby validating its efficacy;
FIG. 14A is a schematic representation of an “operon-like” cluster of novel human hairpin sequences detected by a bioinformatic oligonucleotide detection engine constructed and operative in accordance with a preferred embodiment of the present invention, and non-GAM hairpin sequences used as negative controls thereto;
FIG. 14B is a schematic representation of secondary folding of hairpins of the operon-like cluster of FIG. 14A;
FIG. 14C is a picture of laboratory results demonstrating expression of novel oligonucleotides of FIGS. 14A and 14B and lack of expression of the negative controls, thereby validating efficacy of bioinformatic detection of GAM oligonucleotides and GR polynucleotides detected by a bioinformatic oligonucleotide detection engine, constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 15A is an annotated sequence of EST72223 comprising known human microRNA oligonucleotide MIR98 and novel human oligonucleotide GAM25 PRECURSOR detected by the oligonucleotide detection system of the present invention; and
FIGS. 15B, 15C and 15D are pictures of laboratory results demonstrating laboratory confirmation of expression of known human oligonucleotide MIR98 and of novel bioinformatically-detected human GAM25 RNA respectively, both of FIG. 15A, thus validating the bioinformatic oligonucleotide detection system of the present invention;
FIGS. 16A, 16B and 16C are schematic diagrams which, when taken together, represent methods of designing primers to identify specific hairpin oligonucleotides in accordance with a preferred embodiment of the present invention.
FIG. 17A is a simplified flowchart illustrating construction of a microarray constructed and operative to identify novel oligonucleotides of the present invention, in accordance with a preferred embodiment of the present invention;
FIG. 17B is a simplified block diagram illustrating design of a microarray constructed and operative to identify novel oligonucleotides of the present invention, in accordance with a preferred embodiment of the present invention;
FIG. 17C is a flowchart illustrating a mode of preparation and amplification of a cDNA library in accordance with a preferred embodiment of the present invention;
FIG. 18A is a line graph showing results of detection of known microRNA oligonucleotides and of novel GAM oligonucleotides, using a microarray constructed and operative in accordance with a preferred embodiment of the present invention;
FIG. 18B is a line graph showing specificity of hybridization of a microarray constructed and operative in accordance with a preferred embodiment of the present invention; and
FIG. 18C is a summary table demonstrating detection of known microRNA oligonucleotides using a microarray constructed and operative in accordance with a preferred embodiment of the present invention.

BRIEF DESCRIPTION OF SEQUENCES

A Sequence Listing of genomic sequences of the present invention designated SEQ ID NO:1 through SEQ ID: 4,254,670 is attached to this application, and is hereby incorporated herein. The genomic listing comprises the following nucleotide sequences: nucleotide sequences of 21,916 bacterial and 6,100 human GAM precursors of respective novel oligonucleotides of the present invention; nucleotide sequences of 32,713 bacterial and 11,428 human GAM RNA oligonucleotides of respective novel oligonucleotides of the present invention; and nucleotide sequences of 1,507,219 target gene binding sites of respective novel oligonucleotides of the present invention.

DETAILED DESCRIPTION

Reference is now made to FIG. 1, which is a simplified diagram describing a plurality of novel bioinformatically-detected oligonucleotide of the present invention referred to here as the Genomic Address Messenger (GAM) oligonucleotide, which modulates expression of respective target genes whose function and utility are known in the art.
GAM is a novel bioinformatically detectable regulatory, non-protein-coding, miRNA-like oligonucleotide. The method by which GAM is detected is described with additional reference to FIGS. 1-8.
The GAM PRECURSOR is preferably encoded by a bacterial genome. Alternatively or additionally, the GAM PRECURSOR is preferably encoded by the human genome. The GAM TARGET GENE is a gene encoded by the human genome. Alternatively or additionally, the GAM TARGET GENE is a gene encoded by a bacterial genome.
The GAM PRECURSOR encodes a GAM PRECURSOR RNA. Similar to other miRNA oligonucleotides, the GAM PRECURSOR RNA does not encode a protein.
GAM PRECURSOR RNA folds onto itself, forming GAM FOLDED PRECURSOR RNA, which has a two-dimensional “hairpin” structure. GAM PRECURSOR RNA folds onto itself, forming GAM FOLDED PRECURSOR RNA, which has a two-dimensional “hairpin structure”. As is well-known in the art, this “hairpin structure” is typical of RNA encoded by known miRNA precursor oligonucleotides and is due to the full or partial complementarity of the nucleotide sequence of the first half of an miRNA precursor to the RNA that is encoded by a miRNA oligonucleotide to the nucleotide sequence of the second half thereof.
A complementary sequence is a sequence which is reversed and wherein each nucleotide is replaced by a complementary nucleotide, as is well known in the art (e.g. ATGGC is the complementary sequence of GCCAT).
An enzyme complex designated DICER COMPLEX, an enzyme complex composed of Dicer RNaseIII together with other necessary proteins, cuts the GAM FOLDED PRECURSOR RNA yielding a single-stranded ˜22 nt-long RNA segment designated GAM RNA.
GAM TARGET GENE encodes a corresponding messenger RNA, designated GAM TARGET RNA. As is typical of mRNA of a protein-coding gene, each GAM TARGET RNAs of the present invention comprises three regions, as is typical of mRNA of a protein-coding gene: a 5′ untranslated region, a protein-coding region and a 3′ untranslated region, designated 5′UTR, PROTEIN-CODING and 3′UTR, respectively.
GAM RNA binds complementarily to one or more target binding sites located in the untranslated regions of each of the GAM TARGET RNAs of the present invention. This complementary binding is due to the partial or full complementarity between the nucleotide sequence of GAM RNA and the nucleotide sequence of each of the target binding sites. As an illustration, FIG. 1 shows three such target binding sites, designated BINDING SITE I, BINDING SITE II and BINDING SITE III, respectively. It is appreciated that the number of target binding sites shown in FIG. 1 is only illustrative and that any suitable number of target binding sites may be present. It is further appreciated that although FIG. 1 shows target binding sites only in the 3′UTR region, these target binding sites may instead be located in the 5′UTR region or in both the 3′UTR and 5′UTR regions.
The complementary binding of GAM RNA to target binding sites on GAM TARGET RNA, such as BINDING SITE I, BINDING SITE II and BINDING SITE III, inhibits the translation of each of the GAM TARGET RNAs of the present invention into respective GAM TARGET PROTEIN, shown surrounded by a broken line.
It is appreciated that the GAM TARGET GENE in fact represents a plurality of GAM target genes. The mRNA of each one of this plurality of GAM target genes comprises one or more target binding sites, each having a nucleotide sequence which is at least partly complementary to GAM RNA and which when bound by GAM RNA causes inhibition of translation of the GAM target mRNA into a corresponding GAM target protein.
The mechanism of the translational inhibition that is exerted by GAM RNA on one or more GAM TARGET GENEs may be similar or identical to the known mechanism of translational inhibition exerted by known miRNA oligonucleotides.
The nucleotide sequences of each of a plurality of GAM oligonucleotides that are described by FIG. 1 and their respective genomic sources and genomic locations are set forth in Tables 1-3, hereby incorporated herein.
The nucleotide sequences of GAM PRECURSOR RNAs, and a schematic representation of a predicted secondary folding of GAM FOLDED PRECURSOR RNAs, of each of a plurality of GAM oligonucleotides that are described by FIG. 1 are set forth in Table 4, hereby incorporated herein.
The nucleotide sequences of “diced” GAM RNAs of each of a plurality of GAM oligonucleotides that are described by FIG. 1 are set forth in Table 5, hereby incorporated herein.
The nucleotide sequences of target binding sites, such as BINDING SITE I, BINDING SITE II and BINDING SITE III that are found on GAM TARGET RNAs of each of a plurality of GAM oligonucleotides that are described by FIG. 1, and a schematic representation of the complementarity of each of these target binding sites to each of a plurality of GAM RNAs that are described by FIG. 1 are set forth in Tables 6-7, hereby incorporated herein.
It is appreciated that the specific functions and accordingly the utilities of each of a plurality of GAM oligonucleotides that are described by FIG. 1 are correlated with and may be deduced from the identity of the GAM TARGET GENES inhibited thereby, and whose functions are set forth in Table 8, hereby incorporated herein.
Studies documenting the well known correlations between each of a plurality of GAM TARGET GENEs that are described by FIG. 1 and the known gene functions and related diseases are listed in Table 9, hereby incorporated herein.
The present invention discloses a novel group of bacterial and human oligonucleotides, belonging to the miRNA-like oligonucleotide group, here termed GAM oligonucleotides, for which a specific complementary binding has been determined bioinformatically.
Reference is now made to FIG. 2, which is a simplified block diagram illustrating a bioinformatic oligonucleotide detection system and method constructed and operative in accordance with a preferred embodiment of the present invention.
An important feature of the present invention is a bioinformatic oligonucleotide detection engine 100, which is capable of bioinformatically detecting oligonucleotides of the present invention.
The functionality of the bioinformatic oligonucleotide detection engine 100 includes receiving expressed RNA data 102, sequenced DNA data 104, and protein function data 106; performing a complex process of analysis of this data as elaborated hereinbelow, and based on this analysis provides information, designated by reference numeral 108, identifying and describing features of novel oligonucleotides.
Expressed RNA data 102 comprises published expressed sequence tags (EST) data, published mRNA data, as well as other published RNA data. Sequenced DNA data 104 comprises alphanumeric data representing genomic sequences and preferably including annotations such as information indicating the location of known protein-coding regions relative to the genomic sequences.
Protein function data 106 comprises information from scientific publications e.g. physiological functions of known proteins and their connection, involvement and possible utility in treatment and diagnosis of various diseases.
Expressed RNA data 102 and sequenced DNA data 104 may preferably be obtained from data published by the National Center for Biotechnology Information (NCBI) at the National Institute of Health (NIH) (Jenuth, J. P. (2000). Methods Mol. Biol. 132:301-312(2000), herein incorporated by reference) as well as from various other published data sources. Protein function data 106 may preferably be obtained from any one of numerous relevant published data sources, such as the Online Mendelian Inherited Disease In Man (OMIM™, Hamosh et al., Nucleic Acids Res. 30: 52-55(2002)) database developed by John Hopkins University, and also published by NCBI (2000).
Prior to or during actual detection of bioinformatically-detected group of novel oligonucleotides 108 by the bioinformatic oligonucleotide detection engine 100, bioinformatic oligonucleotide detection engine training & validation functionality 110 is operative. This functionality uses one or more known miRNA oligonucleotides as a training set to train the bioinformatic oligonucleotide detection engine 100 to bioinformatically recognize miRNA-like oligonucleotides, and their respective potential target binding sites. Bioinformatic oligonucleotide detection engine training & validation functionality 110 is further described hereinbelow with reference to FIG. 3.
The bioinformatic oligonucleotide detection engine 100 preferably comprises several modules which are preferably activated sequentially, and are described as follows:
A non-protein-coding genomic sequence detector 112 operative to bioinformatically detect non-protein-coding genomic sequences. The non-protein-coding genomic sequence detector 112 is further described herein below with reference to FIGS. 4A and 4B.
A hairpin detector 114 operative to bioinformatically detect genomic “hairpin-shaped” sequences, similar to GAM FOLDED PRECURSOR RNA (FIG. 1). The hairpin detector 114 is further described herein below with reference to FIGS. 5A and 5B.
A Dicer-cut location detector 116 operative to bioinformatically detect the location on a GAM FOLDED PRECURSOR RNA which is enzymatically cut by DICER COMPLEX (FIG. 1), yielding “diced” GAM RNA. The Dicer-cut location detector 116 is further described herein below with reference to FIGS. 6A-6C.
A target gene binding site detector 118 operative to bioinformatically detect target genes having binding sites, the nucleotide sequence of which is partially complementary to that of a given genomic sequence, such as a nucleotide sequence cut by DICER COMPLEX. The target gene binding site detector 118 is further described hereinbelow with reference to FIGS. 7A and 7B.
A function & utility analyzer, designated by reference numeral 120, is operative to analyze the function and utility of target genes in order to identify target genes which have a significant clinical function and utility. The function & utility analyzer 120 is further described hereinbelow with reference to FIG. 8
According to an embodiment of the present invention, the bioinformatic oligonucleotide detection engine 100 may employ a cluster of 40 personal computers (PCs; XEON®, 2.8 GHz, with 80 GB storage each) connected by Ethernet to eight servers (2-CPU, XEON™ 1.2-2.2 GHz, with ˜200 GB storage each) and combined with an 8-processor server (8-CPU, Xeon 550 Mhz w/8 GB RAM) connected via 2 HBA fiber-channels to an EMC CLARIION™ 100-disks, 3.6 Terabyte storage device. A preferred embodiment of the present invention may also preferably comprise software that utilizes a commercial database software program, such as MICROSOFT ™ SQL Server 2000.
According to a preferred embodiment of the present invention, the bioinformatic oligonucleotide detection engine 100 may employ a cluster of 80 Servers (XEON®, 2.8 GHz, with 80 GB storage each) connected by Ethernet to eight servers (2-CPU, XEON™ 1.2-2.2 GHz, with ˜200 GB storage each) and combined with storage device (Promise Technology Inc., RM8000) connected to an 8-disks, 2 Terabytes total. A preferred embodiment of the present invention may also preferably comprise software that utilizes a commercial database software program, such as MICROSOFT ™ SQL Server 2000. It is appreciated that the abovementioned hardware configuration is not meant to be limiting and is given as an illustration only. The present invention may be implemented in a wide variety of hardware and software configurations.
The present invention discloses 21,916 bacterial and 6,100 human novel oligonucleotides of the GAM group of oligonucleotides, which have been detected bioinformatically and 6,056 bacterial and 430 novel polynucleotides of the GR group of polynucleotides, which have been detected bioinformatically. Laboratory confirmation of bioinformatically predicted oligonucleotides of the GAM group of oligonucleotides, and several bioinformatically predicted polynucleotides of the GR group of polynucleotides, is described hereinbelow with reference to FIGS. 13-15D, FIG. 18 and Table 12.
Reference is now made to FIG. 3, which is a simplified flowchart illustrating operation of a preferred embodiment of the bioinformatic oligonucleotide detection engine training & validation functionality 110 described hereinabove with reference to FIG. 2.
bioinformatic oligonucleotide detection engine training & validation functionality 110 begins by training the bioinformatic oligonucleotide detection engine 100 (FIG. 2) to recognize one or more known miRNA oligonucleotides, as designated by reference numeral 122. This training step comprises hairpin detector training & validation functionality 124, further described hereinbelow with reference to FIG. 5A, Dicer-cut location detector training & validation functionality 126, further described hereinbelow with reference to FIGS. 6A and 6B, and target gene binding site detector training & validation functionality 128, further described hereinbelow with reference to FIG. 7A.
Next, the bioinformatic oligonucleotide detection engine training & validation functionality 110 is operative bioinformatically detect novel oligonucleotides, using bioinformatic oligonucleotide detection engine 100 (FIG. 2), as designated by reference numeral 130. Wet lab experiments are preferably conducted in order to validate expression and preferably function of some samples of the novel oligonucleotides detected by the bioinformatic oligonucleotide detection engine 100, as designated by reference numeral 132. FIGS. 13A-15D, FIG. 18 and Table 12 illustrate examples of wet lab validation of sample novel human oligonucleotides bioinformatically-detected in accordance with a preferred embodiment of the present invention.
Reference is now made to FIG. 4A, which is a simplified block diagram of a preferred implementation of the non-protein-coding genomic sequence detector 112 described hereinabove with reference to FIG. 2. The non-protein-coding genomic sequence detector 112 preferably receives at least two types of published genomic data: Expressed RNA data 102 and sequenced DNA data 104. The expressed RNA data 102 may include, inter alia, EST data, EST clusters data, EST genome alignment data and mRNA data. Sources for expressed RNA data 102 include NCBI dbEST, NCBI UniGene clusters and mapping data, and TIGR gene indices (Kirkness F. and Kerlavage, A. R., Methods Mol. Biol. 69:261-268 (1997)). Sequenced DNA data 104 may include sequence data (FASTA format files), and feature annotations (GenBank file format) mainly from NCBI databases. Based on the abovementioned input data, the non-protein-coding genomic sequence detector 112 produces a plurality of non-protein-coding genomic sequences 136. Preferred operation of the non-protein-coding genomic sequence detector 112 is described hereinbelow with reference to FIG. 4B.
Reference is now made to FIG. 4B, which is a simplified flowchart illustrating a preferred operation of the non-protein-coding genomic sequence detector 112 of FIG. 2. Detection of non-protein-coding genomic sequences 136, generally preferably progresses along one of the following two paths:
A first path for detecting non-protein-coding genomic sequences 136 (FIG. 4A) begins with receipt of a plurality of known RNA sequences, such as EST data. Each RNA sequence is first compared with known protein-coding DNA sequences, in order to select only those RNA sequences which are non-protein-coding, i.e. intergenic or intronic sequences. This can preferably be performed by using one of many alignment algorithms known in the art, such as BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). This sequence comparison preferably also provides localization of the RNA sequence on the DNA sequences.
Alternatively, selection of non-protein-coding RNA sequences and their localization on the DNA sequences can be performed by using publicly available EST cluster data and genomic mapping databases, such as the UNIGENE database published by NCBI or the TIGR database. Such databases, map expressed RNA sequences to DNA sequences encoding them, find the correct orientation of EST sequences, and indicate mapping of ESTs to protein-coding DNA regions, as is well known in the art. Public databases, such as TIGR, may also be used to map an EST to a cluster of ESTs, known in the art as Tentative Human Consensus and assumed to be expressed as one segment. Publicly available genome annotation databases, such as NCBI's GenBank, may also be used to deduce expressed intronic sequences.
Optionally, an attempt may be made to “expand” the non-protein RNA sequences thus found, by searching for transcription start and end signals, respectively upstream and downstream of the location of the RNA on the DNA, as is well known in the art.
A second path for detecting non-protein-coding genomic sequences 136 (FIG. 4A) begins with receipt of DNA sequences. The DNA sequences are parsed into non-protein-coding sequences, using published DNA annotation data, by extracting those DNA sequences which are between known protein-coding sequences. Next, transcription start and end signals are sought. If such signals are found, and depending on their robustness, probable expressed non-protein-coding genomic sequences are obtained. Such approach is especially useful for identifying novel GAM oligonucleotides which are found in proximity to other known miRNA oligonucleotides, or other wet lab validated GAM oligonucleotides. Since, as described hereinbelow with reference to FIG. 9, GAM oligonucleotides are frequently found in clusters; sequences located near known miRNA oligonucleotides are more likely to contain novel GAM oligonucleotides. Optionally, sequence orthology, i.e. sequence conservation in an evolutionary related species, may be used to select genomic sequences having a relatively high probability of containing expressed novel GAM oligonucleotides. It is appreciated that in detecting non-human GAM oligonucleotides of the present invention the bioinformatic oligonucleotide detection engine 100 utilizes the input genomic sequences, without filtering protein-coding regions detected by the non-protein-coding genomic sequence detector 112, hence non-protein-coding genomic sequences 136 refers to GENOMIC SEQUENCES only.
Reference is now made to FIG. 5A, which is a simplified block diagram of a preferred implementation of the hairpin detector 114 described hereinabove with reference to FIG. 2.
The goal of the hairpin detector 114 is to detect hairpin-shaped genomic sequences, similar to those of known miRNA oligonucleotides. A hairpin-shaped genomic sequence is a genomic sequence, having a first half which is at least partially complementary to a second half thereof, which causes the halves to folds onto themselves, thereby forming a hairpin structure, as mentioned hereinabove with reference to FIG. 1.
The hairpin detector 114 (FIG. 2) receives a plurality of non-protein-coding genomic sequences 136 (FIG. 4A). Following operation of hairpin detector training & validation functionality 124 (FIG. 3), the hairpin detector 114 is operative to detect and output hairpin-shaped sequences, which are found in the non-protein-coding genomic sequences 136. The hairpin-shaped sequences detected by the hairpin detector 114 are designated hairpin structures on genomic sequences 138. A preferred mode of operation of the hairpin detector 114 is described hereinbelow with reference to FIG. 5B.
hairpin detector training & validation functionality 124 includes an iterative process of applying the hairpin detector 114 to known hairpin-shaped miRNA precursor sequences, calibrating the hairpin detector 114 such that it identifies a training set of known hairpin-shaped miRNA precursor sequences, as well as other similarly hairpin-shaped sequences. In a preferred embodiment of the present invention, the hairpin detector training & validation functionality 124 trains the hairpin detector 114 and validates each of the steps of operation thereof described hereinbelow with reference to FIG. 5B
The hairpin detector training & validation functionality 124 preferably uses two sets of data: the aforesaid training set of known hairpin-shaped miRNA precursor sequences, such as hairpin-shaped miRNA precursor sequences of 440 miRNA oligonucleotides of H. sapiens, M. musculus, C. elegans, C. Brigssae and D. Melanogaster, annotated in the RFAM database (Griffiths-Jones 2003), and a background set of about 1000 hairpin-shaped sequences found in expressed non-protein-coding human genomic sequences. The background set is expected to comprise some valid, previously undetected hairpin-shaped miRNA-like precursor sequences, and many hairpin-shaped sequences which are not hairpin-shaped miRNA-like precursors.
In a preferred embodiment of the present invention the efficacy of the hairpin detector 114 (FIG. 2) is confirmed. For example, when a similarity threshold is chosen such that 87% of the known hairpin-shaped miRNA precursors are successfully predicted, only 21.8% of the 1000 background set of hairpin-shaped sequences are predicted to be hairpin-shaped miRNA-like precursors.
Reference is now made to FIG. 5B, which is a simplified flowchart illustrating preferred operation of the hairpin detector 114 of FIG. 2. The hairpin detector 114 preferably initially uses a secondary structure folding algorithm based on free-energy minimization, such as the MFOLD algorithm, described in Mathews et al. J. Mol. Biol. 288:911-940 (1999) and Zuker, M. Nucleic Acids Res. 31: 3406-3415 (2003), the disclosure of which is hereby incorporated by reference. This algorithm is operative to calculate probable secondary structure folding patterns of the non-protein-coding genomic sequences 136 (FIG. 4A) as well as the free-energy of each of these probable secondary folding patterns. The secondary structure folding algorithm, such as the MFOLD algorithm (Mathews, 1997; Zuker 2003), typically provides a listing of the base-pairing of the folded shape, i.e. a listing of each pair of connected nucleotides in the sequence.
Next, the hairpin detector 114 analyzes the results of the secondary structure folding patterns, in order to determine the presence and location of hairpin folding structures. The goal of this second step is to assess the base-pairing listing provided by the secondary structure folding algorithm, in order to determine whether the base-pairing listing describes one or more hairpin type bonding pattern. Preferably, sequence segment corresponding to a hairpin structure is then separately analyzed by the secondary structure folding algorithm in order to determine its exact folding pattern and free-energy.
The hairpin detector 114 then assesses the hairpin structures found by the previous step, comparing them to hairpin structures of known miRNA precursors, using various characteristic hairpin structure features such as its free-energy and its thermodynamic stability, the amount and type of mismatched nucleotides and the existence of sequence repeat-elements, number of mismatched nucleotides in positions 18-22 counting from loop, and Percent of G nucleotide. Only hairpins that bear statistically significant resemblance to the training set of hairpin structures of known miRNA precursors, according to the abovementioned parameters, are accepted.
In a preferred embodiment of the present invention, similarity to the training set of hairpin structures of known miRNA precursors is determined using a “similarity score” which is calculated using a multiplicity of terms, where each term is a function of one of the above-mentioned hairpin structure features. The parameters of each function are found heuristically from the set of hairpin structures of known miRNA precursors, as described hereinabove with reference to hairpin detector training & validation functionality 124 (FIG. 3). The selection of the features and their function parameters is optimized so as to achieve maximized separation between the distribution of similarity scores validated miRNA precursor hairpin structures, and the distribution of similarity scores of hairpin structures detected in the background set mentioned hereinabove with reference to FIG. 5B.
In an alternative preferred embodiment of the present invention, the step described in the preceding paragraph may be split into two stages. A first stage implements a simplified scoring method, typically based on thresholding a subset of the hairpin structure features described hereinabove, and may employ a minimum threshold for hairpin structure length and a maximum threshold for free-energy. A second stage is preferably more stringent, and preferably employs a full calculation of the weighted sum of terms described hereinabove. The second stage preferably is performed only on the subset of hairpin structures that survived the first stage.
The hairpin detector 114 also attempts to select hairpin structures whose thermodynamic stability is similar to that of hairpin structures of known miRNA precursors. This may be achieved in various ways. A preferred embodiment of the present invention utilizes the following methodology, preferably comprising three logical steps:
First, the hairpin detector 114 attempts to group hairpin structures into “families” of closely related hairpin structures. As is known in the art, a secondary structure folding algorithm typically provides multiple alternative folding patterns, for a given genomic sequence and indicates the free-energy of each alternative folding pattern. It is a particular feature of the present invention that the hairpin detector 114 preferably assesses the various hairpin structures appearing in the various alternative folding patterns and groups' hairpin structures which appear at identical or similar sequence locations in various alternative folding patterns into common sequence location based “families” of hairpins. For example, all hairpin structures whose center is within 7 nucleotides of each other may be grouped into a “family”. Hairpin structures may also be grouped into a “family” if their nucleotide sequences are identical or overlap to a predetermined degree.
It is also a particular feature of the present invention that the hairpin structure “families” are assessed in order to select only those families which represent hairpin structures that are as thermodynamically stable as those of hairpin structures of known miRNA precursors. Preferably only families which are represented in at least a selected majority of the alternative secondary structure folding patterns, typically 65%, 80% or 100% are considered to be sufficiently stable. Our tests suggest that only about 50% of the hairpin structures, predicted by the MFOLD algorithm with default parameters, are members of sufficiently stable families, comparing to about 90% of the hairpin structures that contain known miRNAs. This percent depends on the size of the fraction that was fold. In an alternative embodiment of the present invention we use fractions of size 1000 nts as preferable size. Different embodiment uses other sizes of genomics sequences, more or less strict demand for representation in the alternative secondary structure folding patterns.
It is an additional particular feature of the present invention that the most suitable hairpin structure is selected from each selected family. For example, a hairpin structure which has the greatest similarity to the hairpin structures appearing in alternative folding patterns of the family may be preferred. Alternatively or additionally, the hairpin structures having relatively low free-energy may be preferred.
Alternatively or additionally considerations of homology to hairpin structures of other organisms and the existence of clusters of thermodynamically stable hairpin structures located adjacent to each other along a sequence may be important in selection of hairpin structures. The tightness of the clusters in terms of their location and the occurrence of both homology and clusters may be of significance.
Reference is now made to FIGS. 6A-6C, which together describe the structure and operation of the Dicer-cut location detector 116, described hereinabove with reference to FIG. 2.
Reference is now made to FIG. 6A, which is a simplified block diagram of a preferred implementation of the Dicer-cut location detector 116. The goal of the Dicer-cut location detector 116 is to detect the location in which the DICER COMPLEX, described hereinabove with reference to FIG. 1, dices GAM FOLDED PRECURSOR RNA, yielding GAM RNA.
The Dicer-cut location detector 116 therefore receives a plurality of hairpin structures on genomic sequences, designated by reference numeral 138 (FIG. 5A), and following operation of Dicer-cut location detector training & validation functionality 126 (FIG. 3), is operative to detect a plurality of Dicer-cut sequences from hairpin structures, designated by reference numeral 140.
Reference is now made to FIG. 6B, which is a simplified flowchart illustrating a preferred implementation of Dicer-cut location detector training & validation functionality 126.
A general goal of the Dicer-cut location detector training & validation functionality 126 is to analyze the Dicer-cut locations of known diced miRNA on respective hairpin-shaped miRNA precursors in order to determine a common pattern in these locations, which can be used to predict Dicer-cut locations on GAM folded precursor RNAs.
The Dicer-cut locations of known miRNA precursors are obtained and studied. Locations of the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by their respective distances from the 5′ end of the corresponding hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by the relationship between their locations and the locations of one or more nucleotides along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by the relationship between their locations and the locations of one or more bound nucleotide pairs along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by the relationship between their locations and the locations of one or more mismatched nucleotide pairs along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by the relationship between their locations and the locations of one or more unmatched nucleotides along the hairpin-shaped miRNA precursor. Additionally or alternatively, locations of the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides are preferably represented by their respective distances from the loop located at the center of the corresponding hairpin-shaped miRNA precursor.
One or more of the foregoing location metrics may be employed in the Dicer-cut location detector training & validation functionality 126. Additionally, metrics related to the nucleotide content of the diced miRNA and/or of the hairpin-shaped miRNA precursor may be employed.
In a preferred embodiment of the present invention, Dicer-cut location detector training & validation functionality 126 preferably employs standard machine learning techniques known in the art of machine learning to analyze existing patterns in a given “training set” of examples. Standard machine learning techniques are capable, to a certain degree, of detecting patterns in examples to which they have not been previously exposed that are similar to those in the training set. Such machine learning techniques include, but are not limited to neural networks, Bayesian Modeling, Bayesian Networks, Support Vector Machines (SVM), Genetic Algorithms, Markovian Modeling, Maximum Likelihood Modeling, Nearest Neighbor Algorithms, Decision Trees and other techniques, as is well-known in the art.
In accordance with an embodiment of the present invention, two or more classifiers or predictors based on the abovementioned machine learning techniques are separately trained on the abovementioned training set, and are used jointly in order to predict the Dicer-cut location. As an example, FIG. 6B illustrates operation of two classifiers, a 3′ end recognition classifier and a 5′ end recognition classifier. Most preferably, the Dicer-cut location detector training & validation functionality 126 implements a “best-of-breed” approach employing a pair of classifiers based on the abovementioned Bayesian Modeling and Nearest Neighbor Algorithms, and accepting only “potential GAM RNAs” that score highly on one of these predictors. In this context, “high scores” means scores that have been demonstrated to have low false positive value when scoring known miRNA oligonucleotides. Alternatively, the Dicer-cut location detector training & validation functionality 126 may implement operation of more or less than two classifiers.
Predictors used in a preferred embodiment of the present invention are further described hereinbelow with reference to FIG. 6C. A computer program listing of a computer program implementation of the Dicer-cut location detector training & validation functionality 126 is enclosed on an electronic medium in computer-readable form, and is hereby incorporated by reference herein.
When evaluated on the abovementioned validation set of 440 published miRNA oligonucleotides using k-fold cross validation (Mitchell, 1997) with k=3, the performance of the resulting predictors is as follows: In 70% of known miRNA oligonucleotides, a 5′ end location is correctly determined by a Support Vector Machine predictor within up to two nucleotides; a Nearest Neighbor (EDIT DISTANCE) predictor achieves 56% accuracy (247/440); and a Two-Phased Predictor that uses Bayesian modeling (TWO PHASED) achieves 80% accuracy (352/440) when only the first phase is used. When the second phase (strand choice) is implemented by a naive Bayesian model, the accuracy is 55% (244/440), and when the K-nearest-neighbor modeling is used for the second phase, 374/440 decisions are made and the accuracy is 65% (242/374). A K-nearest-neighbor predictor (FIRST-K) achieves 61% accuracy (268/440). The accuracies of all predictors are considerably higher on top-scoring subsets of published miRNA oligonucleotides.
Finally, in order to validate the efficacy and accuracy of the Dicer-cut location detector 116, a sample of novel oligonucleotides detected thereby is preferably selected, and validated by wet lab experiments. Laboratory results validating the efficacy of the Dicer-cut location detector 116 are described hereinbelow with reference to FIGS. 13-15D, FIG. 18 and also in the enclosed file Table 12.
Reference is now made to FIG. 6C, which is a simplified flowchart illustrating an operation of a Dicer-cut location detector 116 (FIG. 2), constructed and operative in accordance with a preferred embodiment of the present invention. The Dicer-cut location detector 116 preferably comprises a machine learning computer program module, which is trained to recognize Dicer-cut locations on known hairpin-shaped miRNA precursors, and based on this training, is operable to detect Dicer-cut locations of novel GAM RNA (FIG. 1) on GAM FOLDED PRECURSOR RNA (FIG. 1). In a preferred embodiment of the present invention, the Dicer-cut location module preferably utilizes machine learning algorithms, including but not limited to Support Vector Machine, Bayesian modeling, Nearest Neighbors, and K-nearest-neighbor algorithms that are known in the art.
When initially assessing a novel GAM FOLDED PRECURSOR RNA, each 19-24 nt-long segment thereof is considered to be a potential GAM RNA, because the Dicer-cut location is initially unknown.
For each such potential GAM RNA, the location of its 5′ end or the locations of its 5′ and 3′ ends are scored by at least one recognition classifier or predictor, operating on features such as the following: Locations of the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by their respective distances from the 5′ end of the corresponding hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by the relationship between their locations and the locations of one or more nucleotides along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by the relationship between their locations and the locations of one or more bound nucleotide pairs along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by the relationship between their locations and the locations of one or more mismatched nucleotide pairs along the hairpin-shaped miRNA precursor. Additionally or alternatively, the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by the relationship between their locations and the locations of one or more unmatched nucleotides along the hairpin-shaped miRNA precursor. Additionally or alternatively, locations of the 5′ and/or 3′ ends of the known diced miRNA oligonucleotides, which are preferably represented by their respective distances from the loop located at the center of the corresponding hairpin-shaped miRNA precursor. Additionally or alternatively, metrics related to the nucleotide content of the diced miRNA and/or of the hairpin-shaped miRNA precursor.
In a preferred embodiment of the present invention, the Dicer-cut location detector 116 (FIG. 2) may use a Support Vector Machine predictor.
In another preferred embodiment of the present invention, the Dicer-cut location detector 116 (FIG. 2) preferably employs an “EDIT DISTANCE” predictor, which seeks sequences that are similar to those of known miRNA oligonucleotides, utilizing a Nearest Neighbor algorithm, where a similarity metric between two sequences is a variant of the Edit Distance algorithm (Gusfield, 1997). The EDIT DISTANCE predictor is based on an observation that miRNA oligonucleotides tend to form clusters, the members of which show marked sequence similarity.
In yet another preferred embodiment of the present invention, the Dicer-cut location detector 116 (FIG. 2) preferably uses a “TWO PHASE” predictor, which predicts the Dicer-cut location in two distinct phases: (a) selecting a double-stranded segment of the GAM FOLDED PRECURSOR RNA (FIG. 1) comprising the GAM RNA by naive Bayesian modeling and (b) detecting which strand of the double-stranded segment contains GAM RNA (FIG. 1) by employing either naive or K-nearest-neighbor modeling. K-nearest-neighbor modeling is a variant of the “FIRST-K” predictor described hereinbelow, with parameters optimized for this specific task. The “TWO PHASE” predictor may be operated in two modes: either utilizing only the first phase and thereby producing two alternative Dicer-cut location predictions, or utilizing both phases and thereby producing only one final Dicer-cut location.
In still another preferred embodiment of the present invention, the Dicer-cut location detector 116 preferably uses a “FIRST-K” predictor, which utilizes a K-nearest-neighbor algorithm. The similarity metric between any two sequences is 1-E/L, where L is a parameter, preferably 8-10 and E is the edit distance between the two sequences, taking into account only the first L nucleotides of each sequence. If the K-nearest-neighbor scores of two or more locations on the GAM FOLDED PRECURSOR RNA (FIG. 1) are not significantly different, these locations are further ranked by a Bayesian model, similar to the one described hereinabove.
In accordance with an embodiment of the present invention, scores of two or more of the abovementioned classifiers or predictors are integrated, yielding an integrated score for each potential GAM RNA. As an example, FIG. 6C illustrates an integration of scores from two classifiers, a 3′ end recognition classifier and a 5′ end recognition classifier, the scores of which are integrated to yield an integrated score. Most preferably, the INTEGRATED SCORE of FIG. 6C preferably implements a “best-of-breed” approach employing a pair of classifiers and accepting only “potential GAM RNAs” that score highly on one of the abovementioned “EDIT DISTANCE” or “TWO PHASE” predictors. In this context, “high scores” means scores that have been demonstrated to have low false positive value when scoring known miRNA oligonucleotides. Alternatively, the INTEGRATED SCORE may be derived from operation of more or less than two classifiers.
The INTEGRATED SCORE is evaluated as follows: (a) the “potential GAM RNA” having the highest score is preferably taken to be the most probable GAM RNA, and (b) if the integrated score of this most probable GAM RNA is higher than a pre-defined threshold, then the most probable GAM RNA is accepted as a PREDICTED GAM RNA. Preferably, this evaluation technique is not limited to the highest scoring potential GAM RNA.
In a preferred embodiment of the present invention, PREDICTED GAM RNAs comprising a low complexity nucleotide sequence (e.g., ATATATA) may optionally be filtered out, because there is a high probability that they are part of a repeated element in the DNA, and are therefore not functional, as is known in the art. For each PREDICTED GAM RNA sequence, the number of occurrences of each two nt combination (AA, AT, AC) comprised in that sequence is counted. PREDICTED GAM RNA sequences where the sum of the two most probable combinations is higher than a threshold, preferably 8-10, are filtered out. As an example, when the threshold is set such that 2% of the known miRNA oligonucleotides are filtered out, 30% of the predicted GAM RNAs are filtered out.
Reference is now made to FIG. 7A, which is a simplified block diagram of a preferred implementation of the target gene binding site detector 118 described hereinabove with reference to FIG. 2. The goal of the target gene binding site detector 118 is to detect one or more binding sites located in 3′UTRs of the mRNA of a known gene, such as BINDING SITE I, BINDING SITE II and BINDING SITE III (FIG. 1), the nucleotide sequence of which binding sites is partially or fully complementary to a GAM RNA, thereby determining that the abovementioned known gene is a target gene of the GAM RNA.
The target gene binding site detector 118 (FIG. 2) receives a plurality of Dicer-cut sequences from hairpin structures 140 (FIG. 6A) and a plurality of potential target gene sequences 142, which are derived from sequenced DNA data 104 (FIG. 2).
The target gene binding site detector training & validation functionality 128 (FIG. 3) is operative to train the target gene binding site detector 118 on known miRNA oligonucleotides and their respective target genes and to build a background model for an evaluation of the probability of achieving similar results randomly (P value) for the target gene binding site detector 118 results. The target gene binding site detector training & validation functionality 128 constructs the model by analyzing both heuristically and computationally the results of the target gene binding site detector 118.
Following operation of target gene binding site detector training & validation functionality 128 (FIG. 3), the target gene binding site detector 118 is operative to detect a plurality of potential novel target genes having binding site/s 144, the nucleotide sequence of which is partially or fully complementary to that of each of the plurality of Dicer-cut sequences from hairpin structures 140. Preferred operation of the target gene binding site detector 118 is further described hereinbelow with reference to FIG. 7B.
Reference is now made to FIG. 7B, which is a simplified flowchart illustrating a preferred operation of the target gene binding site detector 118 of FIG. 2.
In an embodiment of the present invention, the target gene binding site detector 118 first compares nucleotide sequences of each of the plurality of Dicer-cut sequences from hairpin structures 140 (FIG. 6A) to the potential target gene sequences 142 (FIG. 7A), such as 3′ side UTRs of known mRNAs, in order to find crude potential matches. This step may be performed using a simple alignment algorithm such as BLAST.
Then, the target gene binding site detector 118 filters these crude potential matches, to find closer matches, which more closely resemble published miRNA oligonucleotide binding sites.
Next, the target gene binding site detector 118 expands the nucleotide sequences of the 3′UTR binding site found by the sequence comparison algorithm (e.g. BLAST or EDIT DISTANCE). A determination is made whether any sub-sequence of the expanded sequence may improve the match. The best match is considered the alignment.
Free-energy and spatial structure are computed for the resulting binding sites. Calculation of spatial structure may be performed by a secondary structure folding algorithm based on free-energy minimization, such as the MFOLD algorithm described in Mathews et al. (J. Mol. Biol. 288: 911-940 (1999)) and Zuker (Nucleic Acids Res. 31: 3406-3415 (2003)), the disclosure of which is hereby incorporated by reference. Free-energy, spatial structure and the above preferences are reflected in scoring. The resulting scores are compared with scores characteristic of known binding sites of published miRNA oligonucleotides, and each binding site is given a score that reflects its resemblance to these known binding sites.
Finally, the target gene binding site detector 118 analyzes the spatial structure of the binding site. Each 3′UTR-GAM oligonucleotide pair is given a score. Multiple binding sites of the same GAM oligonucleotides to a 3′UTR are given higher scores than those that bind only once to a 3′UTR.
In a preferred embodiment of the present invention, performance of the target gene binding site detector 118 may be improved by integrating several of the abovementioned logical steps, using the methodology described hereinbelow.
For each of the Dicer-cut sequence from hairpin structures 140, its starting segment, e.g. a segment comprising the first 8 nts from its 5′ end, is obtained. For each starting segment, all of the 9 nt segments that are highly complementary to the starting segment are calculated. These calculated segments are referred to here as “potential binding site end segments”. In a preferred embodiment of the present invention, for each 8 nt starting segment, the potential binding site end segments are all 9 nt segments whose complementary sequence contains a 7-9 nt sub-sequence that is not different from the starting segment by more than an insertion, deletion or replacement of one nt. Calculation of potential binding site end segments is preferably performed by a pre-processing tool that maps all possible 8 nt segments to their respective 9 nt segments.
Next, the mRNAs 3′UTRs is parsed into all the segments, with the same length as the potential binding site end segments, preferably 9 nt segments, comprised in the 3′UTR. Location of each such segment is noted, stored in a performance-efficient data structure and compared to the potential binding site end segments calculated in the previous step.
The target gene binding site detector 118 then expands the binding site sequence, preferably in the binding site 5′ direction (i.e. immediately upstream), assessing the degree of its alignment to the Dicer-cut sequence from hairpin structures 140. Preferably, an alignment algorithm is implemented which uses specific weighting parameters based on an analysis of known miRNA oligonucleotide binding sites. As an example, it is apparent that a good match of the 3′ end of the binding site is critically important, a match of the 5′ end is less important but can compensate for a small number of mismatches at the 3′ end of the binding site, and a match of the middle portion of the binding site is much less important.
Next, the number of binding sites found in a specific 3′UTR, the degree of alignment of each of these binding sites, and their proximity to each other are assessed and compared to these properties found in known binding sites of published miRNA oligonucleotides. In a preferred embodiment, the fact that many of the known binding sites are clustered is used to evaluate the P value of obtaining a cluster of a few binding sites on the same target gene 3′UTR in the following way. It scans different score thresholds and calculates for each threshold the number and positions of possible binding sites with a score above the threshold. It then gets a P value for each threshold from a preprocessed calculated background matrix, described hereinbelow, and a number and positions of binding sites combination. The output score for each Dicer-cut sequences from hairpin structures 140 and potential target gene sequences 142 is the minimal P value, normalized with the number of threshold trails using a Bernoulli distribution. A preference of low P value pairs is made.
As mentioned hereinabove, for each target gene, a preprocessed calculated background matrix is built. The matrix includes rows for each number of miRNA oligonucleotide binding sites (in the preferred embodiment, the matrix includes 7 rows to accommodate 0 to 6 binding sites), and columns for each different score threshold (in the preferred embodiment, the matrix includes 5 columns for 5 different thresholds). Each matrix cell, corresponding to a specific number of binding sites and thresholds, is set to be the probability of getting equal or higher number binding sites and an equal or higher score using random 22 nt-long sequences with the same nucleotide distribution as known miRNA oligonucleotides (29.5% T, 24.5% A, 25% G and 21% C). Those probabilities are calculated by running the above procedure for 10000 random sequences that preserved the known miRNA nucleotide distribution (these sequence will be also referred to as miRNA oligonucleotide random sequences). The P value can be estimated as the number of random sequences that obeys the matrix cell requirement divided by the total number of random sequences (10000). In the preferred embodiment, 2 matrices are calculated. The P values of the second matrix are calculated under a constraint that at least two of the binding site positions are under a heuristically-determined constant value. The values of the second matrix are calculated without this constraint. The target gene binding site detector 118 uses the second matrix if the binding site positions agree with the constraint. Otherwise, it uses the first. In an alternative embodiment, only one matrix is calculated without any constraint on the binding sites positions.
A test performed using the target gene binding site detector 118 shows that all of the known miRNA oligonucleotide target genes are found using this algorithm with a P value of less than 0.5%. Running known miRNA oligonucleotides against 3400 potential 3′UTR of target gene sequences yields on average 32 target genes for each miRNA oligonucleotide with a P value less than 0.5%, while background sequences, as well as inverse or complement sequence of known miRNA oligonucleotide (which preserve their high order sequence statistics) found, as expected, 17 target genes on average. This result reflects that the algorithm has the ability to detect real target genes with 47% accuracy.
Finally, orthology data may optionally be used to further prefer binding sites based on their conservation. Preferably, this may be used in cases such as (a) where both the target mRNA and miRNA oligonucleotide have orthologues in another organism, e.g. Human-Mouse orthology, or (b) where a miRNA oligonucleotide (e.g. viral miRNA oligonucleotide) targets two mRNAs in orthologous organisms. In such cases, binding sites that are conserved are preferred.
In accordance with another preferred embodiment of the present invention, binding sites may be searched by a reverse process. Sequences of K (preferably 22) nucleotides in a UTR of a target gene are assessed as potential binding sites. A sequence comparison algorithm, such as BLAST or EDIT DISTANCE variant, is then used to search elsewhere in the genome for partially or fully complementary sequences that are found in known miRNA oligonucleotides or computationally-predicted GAM oligonucleotides. Only complementary sequences that meet predetermined spatial structure and free-energy criteria as described hereinabove, are accepted. Clustered binding sites are strongly preferred and potential binding sites and potential GAM oligonucleotides that occur in evolutionarily-conserved genomic sequences are also preferred. Scoring of candidate binding sites takes into account free-energy and spatial structure of the binding site complexes, as well as the aforesaid preferences.
The 3′UTR of each bacterial gene is extracted from the 500 nts that lay downstream to the gene-coding region. Care is taken that the extracted 3′UTR is not partly covered by the predicted 5′UTR of the next gene-coding region, considered 300 nts upstream. This method is applied on known (not hypothetical) bacterial genes of completed pathogenic eubacterial genomes taken from the updated NCBI Ref_seq database on 17 Mar. 2004.
Reference is now made to FIG. 8, which is a simplified flowchart illustrating a preferred operation of the function & utility analyzer 120 described hereinabove with reference to FIG. 2. The goal of the function & utility analyzer 120 is to determine if a potential target gene is in fact a valid clinically useful target gene. Since a potential novel GAM oligonucleotide binding a binding site in the UTR of a target gene is understood to inhibit expression of that target gene, and if that target gene is shown to have a valid clinical utility, then in such a case it follows that the potential novel oligonucleotide itself also has a valid useful function which is the opposite of that of the target gene.
The function & utility analyzer 120 preferably receives as input a plurality of potential novel target genes having binding site/s 144 (FIG. 7A), generated by the target gene binding site detector 118 (FIG. 2). Each potential oligonucleotide is evaluated as follows: First, the system checks to see if the function of the potential target gene is scientifically well established. Preferably, this can be achieved bioinformatically by searching various published data sources presenting information on known function of proteins. Many such data sources exist and are published, as is well known in the art. Next, for those target genes the function of which is scientifically known and is well documented, the system then checks if scientific research data exists which links them to known diseases. For example, a preferred embodiment of the present invention utilizes the OMIM™ (Hamosh et al, 2002) database published by NCBI, which summarizes research publications relating to genes which have been shown to be associated with diseases. Finally, the specific possible utility of the target gene is evaluated. While this process too may be facilitated by bioinformatic means, it might require manual evaluation of published scientific research regarding the target gene, in order to determine the utility of the target gene to the diagnosis and or treatment of specific disease. Only potential novel oligonucleotides, the target genes of which have passed all three examinations, are accepted as novel oligonucleotide.
Reference is now made to FIG. 9, which is a simplified diagram describing each of a plurality of novel bioinformatically-detected regulatory polynucleotide referred to in this Table as the Genomic Record (GR) polynucleotide. GR encodes an operon-like cluster of novel miRNA-like oligonucleotides, each of which in turn modulates expression of at least one target gene. The function and utility of at least one target gene is known in the art.
The GR PRECURSOR is a novel, bioinformatically-detected, regulatory, non-protein-coding polynucleotide. The method by which the GR PRECURSOR is detected is described hereinabove with additional reference to FIGS. 1-9.
GR PRECURSOR is preferably encoded by the bacterial genome and contains a cluster of novel bacterial oligonucleotides, which preferably bind to human target genes or to bacterium genes. Alternatively or additionally, GR PRECURSOR is encoded by the human genome and contains a cluster of novel human oligonucleotides, which preferably bind to bacterial target genes or to human genes.
The GR PRECURSOR encodes GR PRECURSOR RNA that is typically several hundred to several thousand nts long. The GR PRECURSOR RNA folds spatially, forming the GR FOLDED PRECURSOR RNA. It is appreciated that the GR FOLDED PRECURSOR RNA comprises a plurality of what is known in the art as hairpin structures. Hairpin structures result from the presence of segments of the nucleotide sequence of GR PRECURSOR RNA in which the first half of each such segment has a nucleotide sequence which is at least a partial, and sometimes an accurate, reverse-complement sequence of the second half thereof, as is well known in the art.
The GR FOLDED PRECURSOR RNA is naturally processed by cellular enzymatic activity into a plurality of separate GAM precursor RNAs herein schematically represented by GAM1 FOLDED PRECURSOR RNA through GAM3 FOLDED PRECURSOR RNA. Each GAM folded precursor RNA is a hairpin-shaped RNA segment, corresponding to GAM FOLDED PRECURSOR RNA of FIG. 1.
The abovementioned GAM folded precursor RNAs are diced by DICER COMPLEX of FIG. 1, yielding short RNA segments of about 22 nts in length schematically represented by GAM1 RNA through GAM3 RNA. Each GAM RNA corresponds to GAM RNA of FIG. 1. GAM1 RNA, GAM2 RNA and GAM3 RNA each bind complementarily to binding sites located in the untranslated regions of their respective target genes, designated GAM1 TARGET RNA, GAM2 TARGET RNA and GAM3 TARGET RNA, respectively. These target binding sites correspond to BINDING SITE I, BINDING SITE II and BINDING SITE III of FIG. 1. The binding of each GAM RNA to its target RNA inhibits the translation of its respective target proteins, designated GAM1 TARGET PROTEIN, GAM2 TARGET PROTEIN and GAM3 TARGET PROTEIN, respectively.
It is appreciated that the specific functions, and accordingly the utilities, of the GR polynucleotide are correlated with and may be deduced from the identity of the target genes that are inhibited by GAM RNAs that are present in the operon-like cluster of the polynucleotide. Thus, for the GR polynucleotide, schematically represented by GAM1 TARGET PROTEIN through GAM3 TARGET PROTEIN that are inhibited by the GAM RNA. The function of these target genes is elaborated in Table 8, hereby incorporated herein.
Reference is now made to FIG. 10, which is a block diagram illustrating different utilities of oligonucleotide of the novel group of oligonucleotides of the present invention referred to here as GAM oligonucleotides and GR polynucleotides. The present invention discloses a first plurality of novel oligonucleotides referred to here as GAM oligonucleotides and a second plurality of operon-like polynucleotides referred to here as GR polynucleotides, each of the GR polynucleotide encoding a plurality of GAM oligonucleotides. The present invention further discloses a very large number of known target genes, which are bound by, and the expression of which is modulated by each of the novel oligonucleotides of the present invention. Published scientific data referenced by the present invention provides specific, substantial, and credible evidence that the abovementioned target genes modulated by novel oligonucleotides of the present invention, are associated with various diseases. Specific novel oligonucleotides of the present invention, target genes thereof and diseases associated therewith, are described hereinbelow with reference to Tables 1 through 12. It is therefore appreciated that a function of GAM oligonucleotides and GR polynucleotides of the present invention is modulation of expression of target genes related to known bacterial diseases, and that therefore utilities of novel oligonucleotides of the present invention include diagnosis and treatment of the abovementioned diseases.
FIG. 10 describes various types of diagnostic and therapeutic utilities of novel oligonucleotides of the present invention. A utility of novel oligonucleotide of the present invention is detection of GAM oligonucleotides and of GR polynucleotides. It is appreciated that since GAM oligonucleotides and GR polynucleotides modulate expression of disease related target genes, that detection of expression of GAM oligonucleotides in clinical scenarios associated with said bacterial diseases is a specific, substantial and credible utility. Diagnosis of novel oligonucleotides of the present invention may preferably be implemented by RNA expression detection techniques, including but not limited to biochips, as is well known in the art. Diagnosis of expression of oligonucleotides of the present invention may be useful for research purposes, in order to further understand the connection between the novel oligonucleotides of the present invention and the abovementioned related bacterial diseases, for disease diagnosis and prevention purposes, and for monitoring disease progress.
Another utility of novel oligonucleotides of the present invention is anti-GAM therapy, a mode of therapy which allows up regulation of a bacterial disease-related target gene of a novel GAM oligonucleotide of the present invention, by lowering levels of the novel GAM oligonucleotide which naturally inhibits expression of that target gene. This mode of therapy is particularly useful with respect to target genes which have been shown to be under-expressed in association with a specific bacterial disease. Anti-GAM therapy is further discussed hereinbelow with reference to FIGS. 11A and 11B.
A further utility of novel oligonucleotides of the present invention is GAM replacement therapy, a mode of therapy which achieves down regulation of a bacterial disease related target gene of a novel GAM oligonucleotide of the present invention, by raising levels of the GAM which naturally inhibits expression of that target gene. This mode of therapy is particularly useful with respect to target genes which have been shown to be over-expressed in association with a specific bacterial disease. GAM replacement therapy involves introduction of supplementary GAM products into a cell, or stimulation of a cell to produce excess GAM products. GAM replacement therapy may preferably be achieved by transfecting cells with an artificial DNA molecule encoding a GAM which causes the cells to produce the GAM product, as is well known in the art.
Yet a further utility of novel oligonucleotides of the present invention is modified GAM therapy. Disease conditions are likely to exist, in which a mutation in a binding site of a GAM RNA prevents natural GAM RNA to effectively bind inhibit a bacterial disease related target gene, causing up regulation of that target gene, and thereby contributing to the disease pathology. In such conditions, a modified GAM oligonucleotides is designed which effectively binds the mutated GAM binding site, i.e. is an effective anti-sense of the mutated GAM binding site, and is introduced in disease effected cells. Modified GAM therapy is preferably achieved by transfecting cells with an artificial DNA molecule encoding the modified GAM which causes the cells to produce the modified GAM product, as is well known in the art.
Reference is now made to FIGS. 11A and 11B, which are simplified diagrams which when taken together illustrate anti-GAM therapy mentioned hereinabove with reference to FIG. 10. A utility of novel GAMs of the present invention is anti-GAM therapy, a mode of therapy which allows up regulation of a bacterial disease-related target gene of a novel GAM of the present invention, by lowering levels of the novel GAM which naturally inhibits expression of that target gene. FIG. 11A shows a normal GAM inhibiting translation of a target gene by binding of GAM RNA to a BINDING SITE found in an untranslated region of GAM TARGET RNA, as described hereinabove with reference to FIG. 1.
FIG. 11B shows an example of anti-GAM therapy. ANTI-GAM RNA is short artificial RNA molecule the sequence of which is an anti-sense of GAM RNA. Anti-GAM treatment comprises transfecting diseased cells with ANTI-GAM RNA, or with a DNA encoding thereof. The ANTI-GAM RNA binds the natural GAM RNA, thereby preventing binding of natural GAM RNA to its BINDING SITE. This prevents natural translation inhibition of GAM TARGET RNA by GAM RNA, thereby up regulating expression of GAM TARGET PROTEIN.
It is appreciated that anti-GAM therapy is particularly useful with respect to target genes which have been shown to be under-expressed in association with a specific bacterial disease.
Furthermore, anti-GAM therapy is particularly useful, since it may be used in situations in which technologies known in the art as RNAi and siRNA can not be utilized. As in known in the art, RNAi and siRNA are technologies which offer means for artificially inhibiting expression of a target protein, by artificially designed short RNA segments which bind complementarily to mRNA of said target protein. However, RNAi and siRNA can not be used to directly up regulate translation of target proteins.
Reference is now made to FIG. 12A, which is a bar graph illustrating performance results of the hairpin detector 114 (FIG. 2) constructed and operative in accordance with a preferred embodiment of the present invention.
FIG. 12A illustrates efficacy of several features used by the hairpin detector 114 to detect GAM FOLDED PRECURSOR RNAs (FIG. 1). The values of each of these features is compared between a set of published miRNA precursor oligonucleotides, represented by shaded bars, and a set of random hairpins folded from the human genome denoted hereinbelow as a hairpin background set, represented by white bars. The published miRNA precursor oligonucleotides set is taken from RFAM database, Release 2.1 and includes 148 miRNA oligonucleotides from H. Sapiens.The background set comprises a set of 10,000 hairpins folded from the human genome.
It is appreciated that the hairpin background set is expected to comprise some valid, previously undetected hairpin-shaped miRNA precursor-like GAM FOLDED PRECURSOR RNAs of the present invention, and many hairpin-shaped sequences that are not hairpin-shaped miRNA-like precursors.
For each feature, the bars depict the percent of known miRNA hairpin precursors (shaded bars) and the percent of background hairpins (white bars) that pass the threshold for that feature. The percent of known miRNA oligonucleotides that pass the threshold indicates the sensitivity of the feature, while the corresponding background percent implies the specificity of the feature, although not precisely, because the background set comprises both true and false examples.
The first bar pair, labeled Thermodynamic Stability Selection, depicts hairpins that have passed the selection of “families” of closely related hairpin structures, as described hereinabove with reference to FIG. 5B.
The second bar pair, labeled Hairpin Score, depicts hairpins that have been selected by hairpin detector 114 (FIG. 5B), regardless of the “families” selection.
The third bar pair, labeled Conserved, depicts hairpins that are conserved in human, mouse and rat, (UCSC Goldenpath™ HG16 database).
The fourth bar pair, labeled Expressed, depicts hairpins that are found in EST blocks.
The fifth bar pair, labeled Integrated Selection, depicts hairpin structures predicted by a preferred embodiment of the present invention to be valid GAM PRECURSORs. In a preferred embodiment of the present invention, a hairpin may be considered to be a GAM PRECURSOR if its hairpin detector score is above 0, and it is in one of the following groups: a) in an intron and conserved or b) in an intergenic region and conserved or c) in an intergenic region and expressed, as described below. Further filtering of GAM precursor may be obtained by selecting hairpins with a high score of Dicer-cut location detector 116 as described hereinabove with reference to FIGS. 6A-6C, and with predicted miRNA oligonucleotides, which pass the low complexity filter as described hereinabove, and whose targets are selected by the target gene binding site detector 118 as described hereinabove with reference to FIGS. 7A-7B.
It is appreciated that these results validate the sensitivity and specificity of the hairpin detector 114 (FIG. 2) in identifying novel GAM FOLDED PRECURSOR RNAs, and in effectively distinguishing them from the abundant hairpins found in the genome.
Reference is now made to FIG. 12B, which is a line graph illustrating accuracy of a Dicer-cut location detector 116 (FIG. 2) constructed and operative in accordance with a preferred embodiment of the present invention.
To determine the accuracy of the Dicer-cut location detector 116, a stringent training and test set was chosen from the abovementioned set of 440 known miRNA oligonucleotides, such that no two miRNA oligonucleotides in the set are homologous. This was performed to get a lower bound on the accuracy and avoid effects of similar known miRNA oligonucleotides appearing in both the training and test sets. On this stringent set of size 204, mfold cross validation with k=3 was performed to determine the percent of known miRNA oligonucleotides in which the Dicer-cut location detector 116 described hereinabove predicted the correct miRNA oligonucleotide up to two nucleotides from the correct location. The accuracy of the TWO PHASED predictor is depicted in the graph. The accuracy of the first phase of the TWO PHASED predictor is depicted by the upper line, and that of both phases of the TWO PHASED predictor is depicted by the lower line. Both are binned by the predictor score, where the score is the score of the first stage.
It is appreciated that these results validate the accuracy of the Dicer-cut location detector 116.
Reference is now made to FIG. 12C, which is a bar graph illustrating the performance results of the target gene binding site detector 118 (FIG. 7A) constructed and operative in accordance with a preferred embodiment of the present invention.
FIG. 12C illustrates specificity and sensitivity of the target gene binding site detector 118. The values presented are the result of testing 10000 artificial miRNA oligonucleotide sequences (random 22 nt sequences with the same base composition as published miRNA oligonucleotide sequence). Adjusting the threshold parameters to fulfill 90% sensitivity of validated, published miRNA-3′UTR pairs, requires the P VAL of potential target gene sequences-Dicer-cut sequences to be less than 0.01 and also the P VAL of potential target ortholog gene sequences-Dicer-cut sequences to be less than 0.05. The target gene binding site detector 118 can filter out 99.7% of potential miRNA/gene pairs, leaving only the 0.3% that contain the most promising potential miRNA/gene pairs. Limiting the condition for the P VAL of potential target ortholog gene sequences-Dicer-cut sequences to be less than 0.01 reduces the sensitivity ratio to 70% but filters out more then 50% of the remaining 0.3%, to a final ratio of less than 0.15%.
It is appreciated that these results validate the sensitivity and specificity of the target gene binding site detector 118.
Reference is now made to FIG. 13, which is a summary table of laboratory results validating the expression of 29 novel human GAM RNA oligonucleotides in HeLa cells or, alternatively, in liver or thymus tissues detected by the bioinformatic oligonucleotide detection engine 100 (FIG. 2).
As a positive control, we used a reference set of eight known human miRNA oligonucleotides: hsa-MIR-21; hsa-MIR-27b; hsa-MIR-186; hsa-MIR-93; hsa-MIR-26a; hsa-MIR-191; hsa-MIR-31; and hsa-MIR-92. All positive controls were successfully validated by sequencing.
The table of FIG. 13 lists all GAM RNA predictions whose expression was validated. The field “Primer Sequence” contains the “specific” part of the primer; the field “Sequenced sequence” represents the nucleotide sequence detected by cloning (excluding the hemispecific primer sequence); the field “Predicted GAM RNA” contains the GAM RNA predicted sequence; the field “Distance indicate the distance from Primer; the number of mismatches between the “specific” region of the primer and the corresponding part of the GAM RNA sequence; the field “GAM Name” contains GAM RNA PRECURSOR ID followed by “A” or “B”, which represents the GAM RNA position on the precursor as elaborated in the attached Tables.
A primer was designed such that its first half, the 5′ region, is complementary to the adaptor sequence and its second half, the 3′ region, anneals to the 5′ terminus of GAM RNA sequence, yielding a hemispecific primer (as elaborated hereinbelow in the Methods section). A sample of 13 predicted GAM RNA sequences was examined by PCR using hemispecific primers and a primer specific to the 3′ adaptor. PCR products were cloned into plasmid vectors and then sequenced. For all 13 predicted GAM RNA sequences, the GAM RNA sequence found in the hemispecific primer plus the sequence observed between the hemispecific primer and the 3′ adaptor was completely included in the expected GAM RNA sequence (rows 1-7, and 29). The rest are GAM RNA predictions that were verified by cloning and sequencing, yet, by using a primer that was originally designed for a slightly different prediction.
It is appreciated that failure to detect a predicted oligonucleotide in the lab does not necessarily indicate a mistaken bioinformatic prediction. Rather, it may be due to technical sensitivity limitation of the lab test, or because the predicted oligonucleotides are not expressed in the tissue examined, or at the development phase tested. The observed GAM RNAs may be strongly expressed in HeLa cells while the original GAM RNAs are expressed at low levels in HeLa cells or not expressed at all. Under such circumstances, primer sequences containing up to three mismatches from a specific GAM RNA sequence may amplify it. Thus, we also considered cases in which differences of up to 3 mismatches in the hemispecific primer occur.
The 3′ terminus of observed GAM RNA sequences is often truncated or extended by one or two nucleotides. Cloned sequences that were sequenced from both 5′ and 3′ termini have an asterisk appended to the row number.
Interestingly, the primer sequence followed by the observed cloned sequence is contained within five GAM RNA sequences of different lengths, and belong to 24 precursors derived from distinct loci (Row 29). Out of these, one precursor appears four times in the genome and its corresponding GAM Names are 351973-A, 352169-A, 352445-A and 358164-A.
The sequence presented in Row 29 is a representative of the group of five GAM RNAs. The full list of GAM RNA sequences and their corresponding precursors is as follows (each GAM RNA sequence is followed by the GAM Name): TCACTGCAACCTCCACCTCCCA (352092, 352651, 355761), TCACTGCAACCTCCACCTCCCG (351868, 352440, 351973, 352169, 352445, 358164, 353737, 352382, 352235, 352232, 352268, 351919, 352473, 352444, 353638, 353004, 352925, 352943), TCACTGCAACCTCCACCTCCTG (358311), TCACTGCAACCTCCACCTTCAG (353323), and TCACTGCAACCTCCACCTTCCG (353856).
Method Section
Cell Lines
Three common human cell lines, obtained from Dr. Yonat Shemer at Soroka Medical Center, Be'er Sheva, Israel, were used for RNA extraction; Human Embryonic Kidney HEK-293 cells, Human Cervix Adenocarcinoma HeLa cells and Human Prostate Carcinoma PC3 cells.
RNA Purification
Several sources of RNA were used to prepare libraries:
Total HeLa S100 RNA was prepared from HeLa S100 cellular fraction (4 C Biotech, Belgium) through an SDS (1%)-Proteinase K (200 g/ml) 30 minute incubation at 37 C followed by an acid Phenol-Chloroform purification and isopropanol precipitation (Sambrook et al; Molecular Cloning-A Laboratory Manual).
Total HeLa, HEK-293 and PC3 cell RNA was prepared using the standard Tri-Reagent protocol (Sigma) according to the manufacturer's instructions, except that 1 volume of isopropanol was substituted with 3 volumes of ethanol.
Nuclear and Cytoplasmic RNA was prepared from HeLa or HEK-293 cells in the following manner:
Cell were washed and harvested in ice-cold PBS and precipitated in a swing-out rotor at 1200 rpm at 4 C for 5 minutes. Pellets were loosened by gentle vortexing. 4 ml of “NP40 lysis buffer” (10 mM Tris HCl, 5 mM MgCl2, 10 mM NaCl, 0.5% Nonidet P40, 1 mM Spermidine, 1 mM DTT, 140 U/ml rRnasine) was then added per 5*107 cells. Cells and lysis buffer were incubated for 5 minutes on ice and centrifuged in a swing-out rotor at 500×g at 4 C for 5 minutes. Supernatant, termed cytoplasm, is carefully removed to a tube containing SDS (1% final) and proteinase-K (200 g/ml final). Pellet, termed nuclear fraction, is re-washed and incubated with a similar amount of fresh lysis buffer. Lysis is monitored visually under a microscope at this stage, typically for 5 minutes. Nuclei are pelleted in a swing-out rotor at 500×g at 4 C for 5 minutes. Supernatant is pooled, incubated at 37 C for 30 minutes, Phenol/Chloroform-extracted, and RNA is alcohol-precipitated (Sambrook et al). Nuclei are loosened and then homogenized immediately in >10 volumes of Tri-Reagent (Sigma). Nuclear RNA is then prepared according to the manufacturer's instructions.
Total Tissue RNA
Total tissue RNA was obtained from Ambion USA, and included Human Liver, Thymus, Placenta, Testes and Brain.
RNA Size Fractionation
RNA used for libraries was always size-fractionated. Fractionation was done by loading up to 500 microgram RNA per YM100 Amicon Microcon column (Millipore) followed by a 500×g centrifugation for 40 minutes at 4 C. Flow-through “YM100” RNA is about one quarter of the total RNA and was used for library preparation or fractionated further by loading onto a YM30 Amicon Microcon column (Millipore) followed by a 13,500×g centrifugation for 25 minutes at 4 C. Flow-through “YM30” was used for library preparation “as is” and consists of less than 0.5% of total RNA. Additional size fractionation was achieved during library preparation.
Library Preparation
Two types of cDNA libraries, designated “One-tailed” and “Ligation”, were prepared from the one of the abovementioned fractionated RNA samples. RNA was dephosphorylated and ligated to an RNA (designated with lowercase letters)-DNA (designated with UPPERCASE letters) hybrid 5′-phosphorylated, 3′ idT blocked 3′-adapter (5′-P-uuuAACCGCATCCTTCTC-idT-3′ Dharmacon # P-002045-01-05) (as elaborated in Elbashir et al., Genes Dev. 15:188-200 (2001)) resulting in ligation only of RNase III type cleavage products. 3′-Ligated RNA was excised and purified from a half 6%, half 13% polyacrylamide gel to remove excess adapter with a Nanosep 0.2 microM centrifugal device (Pall) according to instructions, and precipitated with glycogen and 3 volumes of ethanol. Pellet was resuspended in a minimal volume of water.
For the “Ligation” library, a DNA (UPPERCASE)-RNA (lowercase) hybrid 5′-adapter
(5′-TACTAATACGACTCACTaaa-3′ Dharmacon # P-002046-01-05) was ligated to the 3′-adapted RNA, reverse transcribed with “EcoRI-RT”:
(5′-GACTAGCTGGAATTCAAGGATGCGGTTAAA-3′), PCR-amplified with two external primers essentially as in Elbashir et al. (2001), except that primers were “EcoRI-RT” and “PstI
Fwd”(5′-CAGCCAACGCTGCAGATACGACTCACTAAA-3′). This PCR product was used as a template for a second round of PCR with one hemispecific and one external primer or with two hemispecific primers.
For the “One-tailed” library, the 3′-adapted RNA was annealed to 20 pmol primer “EcoRI RT” by heating to 70 C and cooling 0.1 C/sec to 30 C and then reverse-transcribed with Superscript II RT (according to manufacturer's instructions, Invitrogen) in a 20 microliters volume for 10 alternating 5 minute cycles of 37 C and 45 C. Subsequently, RNA was digested with 1 microliter 2M NaOH and 2 mM EDTA at 65 C for 10 minutes. cDNA was loaded on a polyacrylamide gel, excised and gel-purified from excess primer as above (invisible, judged by primer run alongside) and resuspended in 13 microliters of water. Purified cDNA was then oligo-dC tailed with 400 U of recombinant terminal transferase (Roche Molecular Biochemicals), 1 microliter 100 microM dCTP, 1 microliter 15 mM CoCl2, and 4 microliters reaction buffer, to a final volume of 20 microliters for 15 minutes at 37 C. Reaction was stopped with 2 microliters 0.2M EDTA and 15 microliters 3M NaOAc pH 5.2. Volume was adjusted to 150 microliters with water, Phenol: Bromochloropropane 10:1 extracted and subsequently precipitated with glycogen and 3 volumes of ethanol. C-tailed cDNA was used as a template for PCR with the external primers
“T3-PstBsg (G/I) 18”(5′-AATTAACCCTCACTAAAGGCTGCAG GTGCAGGIGGGIIGGGIIGGGIIGN-3′ where I stands for Inosine and N for any of the 4 possible deoxynucleotides), and with “EcoRI
Nested”(5′-GGAATTCAAGGATGCGGTTA-3′). This PCR product was used as a template for a second round of PCR with one hemispecific and one external primer or with two hemispecific primers.
Primer Design and PCR
Hemispecific primers were constructed for each predicted GAM RNA oligonucleotide by an in-house program designed to choose about half of the 5′ or 3′ sequence of the GAM RNA corresponding to a TM of about 30-34 C constrained by an optimized 3′ clamp, appended to the cloning adapter sequence (for “One-tailed” libraries, 5′-GGNNGGGNNG on the 5′ end or TTTAACCGCATC-3′ on the 3′ end of the GAM RNA; for “Ligation” libraries, the same 3′ adapter and 5′-CGACTCACTAAA on the 5′ end of the GAM RNA). Consequently, a fully complementary primer of a TM higher than 60 C was created covering only one half of the GAM RNA sequence permitting the unbiased elucidation by sequencing of the other half.
For each primer, the following criteria were used: Primers were graded according to the TM of the primer half and the nucleotide content of 3 nucleotides of the 3′ clamp from worst to best, roughly: GGG-3′<CCC-3′<TTT-3′/AAA-3′<GG-3′<CC-3′<a TM lower than 30<a TM higher than 34<TT-3′/AA-3′<3G/C nucleotide combination <3 A/T nucleotide combination <any combination of two/three different nucleotides <any combination of three/three different nucleotides.
Validation PCR Product by Southern Blot
GAM RNA oligonucleotides were validated by hybridization of Polymerase Chain Reaction (PCR)-product Southern blots with a probe to the predicted GAM RNA.
PCR product sequences were confirmed by Southern blot (Southern E. M., Biotechnology 1992, 24:122-139 (1975)) and hybridization with DNA oligonucleotide probes synthesized as complementary (antisense) to predicted GAM RNA oligonucleotides. Gels were transferred onto a Biodyne PLUS 0.45 m (Pall) positively charged nylon membrane and UV cross-linked. Hybridization was performed overnight with DIG-labeled probes at 42 C in DIG Easy-Hyb buffer (Roche). Membranes were washed twice with 2×SSC and 0.1% SDS for 10 minutes at 42 C and then washed twice with 0.5×SSC and 0.1% SDS for 5 min at 42 C. The membrane was then developed by using a DIG luminescent detection kit (Roche) using anti-DIG and CSPD reaction, according to the manufacturer's protocol. All probes were prepared according to the manufacturer's (Roche Molecular Biochemicals) protocols: Digoxigenin (DIG) labeled antisense transcripts were prepared from purified PCR products using a DIG RNA labeling kit with T3 RNA polymerase. DIG-labeled PCR was prepared by using a DIG PCR labeling kit. 3′-DIG-tailed oligo ssDNA anti-sense probes, containing DIG-dUTP and dATP at an average tail length of 50 nts were prepared from 100 pmole oligonucleotides with the DIG Oligonucleotide Labeling Kit. Control reactions contained all of the components of the test reaction except library template.
Validation of PCR Product by Nested PCR on the Ligation
To further validate predicted GAM PCR product sequence derived from hemi-primers, a PCR-based diagnostic technique was devised to amplify only those products containing at least two additional nucleotides of the non hemi-primer defined part of the predicted GAM RNA oligonucleotide. In essence, a diagnostic primer was designed so that its 3′ end, which is the specificity determining side, was identical to the desired GAM RNA oligonucleotide, 2-10 nts (typically 4-7, chosen for maximum specificity) further into its 3′ end than the nucleotide stretch primed by the hemi-primer. The hemi-primer PCR product was first ligated into a T-cloning vector (pTZ57/T or pGEM-T) as described hereinabove. The ligation reaction mixture was used as template for the diagnostic PCR under strict annealing conditions with the new diagnostic primer in conjunction with a general plasmid-homologous primer, resulting in a distinct ˜200 base-pair product. This PCR product can be directly sequenced, permitting the elucidation of the remaining nucleotides up to the 3′ of the mature GAM RNA oligonucleotide adjacent to the 3′ adapter. Alternatively, following analysis of the diagnostic PCR reaction on an agarose gel, positive ligation reactions (containing a band of the expected size) were transformed into E. coli. Using this same diagnostic technique and as an alternative to screening by Southern blot colony hybridization, transformed bacterial colonies were screened by colony-PCR (Gussow, D. and Clackson, T, Nucleic Acids Res. 17:4000 (1989)) with the nested primer and the vector primer, prior to plasmid purification and sequencing.
Validation of PCR Product by Cloning and Sequencing
PCR products were inserted into pGEM-T (Promega) or pTZ57/T (MBI Fermentas), heat-shock transformed into competent JM109 E. coli (Promega) and seeded on LB-Ampicilin plates with IPTG and Xgal. White and light blue colonies were transferred to duplicate gridded plates, one of which was blotted onto a membrane (Biodyne Plus, Pall) for hybridization with DIG tailed oligo probes (according to instructions, Roche) complementary to the expected GAM. Plasmid DNA from positive colonies was sequenced.
It is appreciated that the results summarize in FIG. 13 validate the efficacy of the bioinformatic oligonucleotide detection engine 100 of the present invention.
Reference is now made to FIG. 14A, which is a schematic representation of a novel human GR polynucleotide, located on chromosome 9, comprising 2 known human miRNA oligonucleotides—MIR24 and MIR23, and 2 novel GAM oligonucleotides, herein designated GAM7617 and GAM252 (later discovered by other researchers as hsa-mir-27b), all marked by solid black boxes. FIG. 14A also schematically illustrates 6 non-GAM hairpin sequences, and one non-hairpin sequence, all marked by white boxes, and serving as negative controls. By “non-GAM hairpin sequences” is meant sequences of a similar length to known miRNA precursor sequences, which form hairpin secondary folding pattern similar to miRNA precursor hairpins, and yet which are assessed by the bioinformatic oligonucleotide detection engine 100 not to be valid GAM PRECURSOR hairpins. It is appreciated that FIG. 14A is a simplified schematic representation, reflecting only the order in which the segments of interest appear relative to one another, and not a proportional distance between the segments.
Reference is now made to FIG. 14B, which is a schematic representation of secondary folding of each of the MIRs and GAMs of the GR MIR24, MIR23, GAM7617 and GAM252, and of the negative control non-GAM hairpins, herein designated N2, N3, N252, N4, N6 and N7. N0 is a non-hairpin control, of a similar length to that of known miRNA precursor hairpins. It is appreciated that the negative controls are situated adjacent to and in between real miRNA oligonucleotides and GAM predicted oligonucleotides and demonstrates similar secondary folding patterns to that of known MIRs and GAMs.
Reference is now made to FIG. 14C, which is a picture of laboratory results of a PCR test upon a YM100 size-fractionated “ligation” library, utilizing a set of specific primer pairs located directly inside the boundaries of the hairpins. Due to the nature of the library the only PCR amplifiable products can result from RNaseIII type enzyme cleaved RNA, as expected for legitimate hairpin precursors presumed to be produced by DROSHA (Lee et al, Nature 425 415-419, 2003). FIG. 14C demonstrates expression of hairpin precursors of known miRNA oligonucleotides hsa-mir23 and hsa-mir24, and of novel bioinformatically-detected GAM7617 and GAM252 hairpins predicted bioinformatically by a system constructed and operative in accordance with a preferred embodiment of the present invention. FIG. 14C also shows that none of the 7 controls (6 hairpins designated N2, N3, N23, N4, N6 and N7 and 1 non-hairpin sequence designated N0) were expressed. N252 is a negative control sequence partially overlapping GAM252.
In the picture, test lanes including template are designated “+” and the control lane is designated “−”. The control reaction contained all the components of the test reaction except library template. It is appreciated that for each of the tested hairpins, a clear PCR band appears in the test (“+”) lane, but not in the control (“−”) lane.
FIGS. 14A through 14C, when taken together validate the efficacy of the bioinformatic oligonucleotide detection engine in: (a) detecting known miRNA oligonucleotides; (b) detecting novel GAM PRECURSOR hairpins which are found adjacent to these miRNA oligonucleotides, and which despite exhaustive prior biological efforts and bioinformatic detection efforts, went undetected; (c) discerning between GAM (or MIR) PRECURSOR hairpins, and non-GAM hairpins.
It is appreciated that the ability to discern GAM-hairpins from non-GAM-hairpins is very significant in detecting GAM oligonucleotides since hairpins are highly abundant in the genome. Other miRNA prediction programs have not been able to address this challenge successfully.
Reference is now made to FIG. 15A, which is an annotated sequence of an EST comprising a novel GAM oligonucleotides detected by the oligonucleotide detection system of the present invention. FIG. 15A shows the nucleotide sequence of a known human non-protein-coding EST (Expressed Sequence Tag), identified as EST72223. The EST72223 clone obtained from TIGR database (Kirkness and Kerlavage, 1997) was sequenced to yield the above 705 bp transcript with a polyadenyl tail. It is appreciated that the sequence of this EST comprises sequences of one known miRNA oligonucleotide, identified as hsa-MIR98, and of one novel GAM oligonucleotide referred to here as GAM25, detected by the bioinformatic oligonucleotide detection engine 100 (FIG. 2) of the present invention.
The sequences of the precursors of the known MIR98 and of the predicted GAM25 precursors are marked in bold, the sequences of the established miRNA 98 and of the predicted miRNA-like oligonucleotide GAM25 are underlined.
Reference is now made to FIGS. 15B, 15C and 15D, which are pictures of laboratory results, which when taken together demonstrate laboratory confirmation of expression of the bioinformatically-detected novel oligonucleotide of FIG. 15A. In two parallel experiments, an enzymatically synthesized capped, EST72223 RNA transcript, was incubated with Hela S100 lysate for 0 minutes, 4 hours and 24 hours. RNA was subsequently harvested, run on a denaturing polyacrylamide gel, and reacted with either a 102 nt antisense MIR98 probe or a 145 nt antisenseGAM25 precursor transcript probe respectively. The Northern blot results of these experiments demonstrated processing of EST72223 RNA by Hela lysate (lanes 2-4, in FIGS. 15B and 15C), into ˜80 bp and ˜22 bp segments, which reacted with the MIR98 precursor probe (FIG. 15B), and into ˜100 bp and ˜24 bp segments, which reacted with the GAM25 precursor probe (FIG. 15C). These results demonstrate the processing of EST72223 by Hela lysate into MIR98 precursor and GAM25 precursor. It is also appreciated from FIG. 15C (lane 1) that Hela lysate itself reacted with the GAM25 precursor probe, in a number of bands, including a ˜100 bp band, indicating that GAM25-precursor is endogenously expressed in Hela cells. The presence of additional bands, higher than 100 bp in lanes 5-9 probably corresponds to the presence of nucleotide sequences in Hela lysate, which contain the GAM25 sequence.
In addition, in order to demonstrate the kinetics and specificity of the processing of MIR98 and GAM25 precursors into their respective mature, “diced” segments, transcripts of MIR98 and of the bioinformatically predicted GAM25 precursors were similarly incubated with Hela S100 lysate, for 0 minutes, 30 minutes, 1 hour and 24 hours, and for 24 hours with the addition of EDTA, added to inhibit Dicer activity, following which RNA was harvested, run on a polyacrylamide gel and reacted with MIR98 and GAM25 precursor probes. Capped transcripts were prepared for in vitro RNA cleavage assays with T7 RNA polymerase, including a m7G (5′) ppp (5′) G-capping reaction using the T7-mMessage mMachine kit (Ambion). Purified PCR products were used as template for the reaction. These were amplified for each assay with specific primers containing a T7 promoter at the 5′ end and a T3 RNA polymerase promoter at the 3′ end. Capped RNA transcripts were incubated at 30 C in supplemented, dialysis concentrated, Hela S100 cytoplasmic extract (4C Biotech, Seneffe, Belgium). The Hela S100 was supplemented by dialysis to a final concentration of 20 mM Hepes, 100 mM KCl, 2.5 mM MgCl2, 0.5 mM DTT, 20% glycerol and protease inhibitor cocktail tablets (Complete mini Roche Molecular Biochemicals). After addition of all components, final concentrations were 100 mM capped target RNA, 2 mM ATP, 0.2 mM GTP, 500 U/ml RNasin, 25 microgram/ml creatine kinase, 25 mM creatine phosphate, 2.5 mM DTT and 50% S100 extract. Proteinase K, used to enhance Dicer activity (Zhang et al., EMBO J. 21, 5875-5885 (2002)) was dissolved in 50 mM Tris- HCl pH 8, 5 mM CaCl2, and 50% glycerol, was added to a final concentration of 0.6 mg/ml. Cleavage reactions were stopped by the addition of 8 volumes of proteinase K buffer 200 Mm Tris-Hcl, pH 7.5, 25 m M EDTA, 300 mM NaCl, and 2% SDS) and incubated at 65 C for 15 min at different time points (0, 0.5, 1, 4, 24 h) and subjected to phenol/chloroform extraction. Pellets were dissolved in water and kept frozen. Samples were analyzed on a segmented half 6%, half 13% polyacrylamide 1XTBE-7M Urea gel.
The Northern blot results of these experiments demonstrated an accumulation of a ˜22 bp segment which reacted with the MIR98 precursor probe, and of a ˜24 bp segment which reacted with the GAM25 precursor probe, over time (lanes 5-8). Absence of these segments when incubated with EDTA (lane 9), which is known to inhibit Dicer enzyme (Zhang et al., 2002), supports the notion that the processing of MIR98 and GAM25 precursors into their “diced” segments is mediated by Dicer enzyme, found in Hela lysate. Other RNases do not utilize divalent cations and are thus not inhibited by EDTA. The molecular sizes of EST72223, MIR-98 and GAM25 and their corresponding precursors are indicated by arrows.
FIG. 15D present Northern blot results of same above experiments with GAM25 probe (24 nt). The results clearly demonstrated the accumulation of mature GAM25 oligonucleotide after 24 h.
To validate the identity of the band shown by the lower arrow in FIGS. 15C and 15D, a RNA band parallel to a marker of 24 base was excised from the gel and cloned as in Elbashir et al (2001) and sequenced. Ninety clones corresponded to the sequence of mature GAM25 oligonucleotide, three corresponded to GAM25* (the opposite arm of the hairpin with a 1-3 nt 3′ overhang) and two to the hairpin-loop.
GAM25 was also validated endogenously by sequencing from both sides from a HeLa YM100 total-RNA “ligation” libraries, utilizing hemispecific primers as described in FIG. 13.
Taken together, these results validate the presence and processing of a novel miRNA-like oligonucleotide, GAM25, which was predicted bioinformatically. The processing of this novel GAM oligonucleotide product, by Hela lysate from EST72223, through its precursor, to its final form was similar to that observed for known miRNA oligonucleotide, MIR98.
Transcript products were 705 nt (EST72223), 102 nt (MIR98 precursor), 125 nt (GAM25 precursor) long. EST72223 was PCR-amplified with T7-EST 72223 forward primer:
5′-TAATACGACTCACTATAGGCCCTTATTAGAGGATTCTGCT -3′ and T3-EST72223 reverse primer:″-AATTAACCCTCACTAAAGGTTTITITTTCCTGAGA CAGAGT-3′. MIR98 was PCR-amplified using EST72223 as a template with T7MIR98 forward primer:
5′-TAATACGACTCACTATAGGGTGAGGTAGTAAGTTGTATT GTT-3′and T3MIR98 reverse primer:
5′-AATTAACCCTCACTAAAGGGAAAGTAGTAAGTTGTATAG TT-3′. GAM25 was PCR-amplified using EST72223 as a template with GAM25 forward primer:
5′-GAGGCAGGAGAATTGCTTGA-3′ and T3-EST72223 reverse primer: 5′-AATTAACCCTCACTAAAGGCCTGAGACAGAGTCT TGCTC-3′.
It is appreciated that the data presented in FIGS. 15A, 15B, 15C and 15D when taken together validate the function of the bioinformatic oligonucleotide detection engine 100 of FIG. 2. FIG. 15A shows a novel GAM oligonucleotide bioinformatically-detected by the bioinformatic oligonucleotide detection engine 100, and FIGS. 15C and 15D show laboratory confirmation of the expression of this novel oligonucleotide. This is in accord with the engine training and validation methodology described hereinabove with reference to FIG. 2.
Reference is now made to FIGS. 16A-C, which schematically represent three methods that are employed to identify GAM FOLDED PRECURSOR RNA from libraries. Each method involves the design of specific primers for PCR amplification followed by sequencing. The libraries include hairpins as double-stranded DNA with two different adaptors ligated to their 5′ and 3′ ends.
Reference is now made to FIG. 16A, which depicts a first method that uses primers designed to the stems of the hairpins. Since the stem of the hairpins often has bulges, mismatches, as well as G-T pairing, which is less significant in DNA than is G-U pairing in the original RNA hairpin, the primer pairs were engineered to have the lowest possible match to the other strand of the stem. Thus, the F-Stem primer, derived from the 5′ stem region of the hairpin, was chosen to have minimal match to the 3′ stem region of the same hairpin. Similarly, the R-stem primer, derived from the 3′ region of the hairpin (reverse complementary to its sequence), was chosen to have minimal match to the 5′ stem region of the same hairpin. The F-Stem primer was extended in its 5′ sequence with the T3 primer (5′-ATTAACCCTCACTAAAGGGA-3′) and the R-Stem primer was extended in its 5′ sequence with the T7 primer (5′-TAATACGACTCACTATAGGG). The extension is needed to obtain a large enough fragment for direct sequencing of the PCR product. Sequence data from the amplified hairpins is obtained in two ways. One way is the direct sequencing of the PCR products using the T3 primer that matches the extension of the F-Stem primer. Another way is the cloning of the PCR products into a plasmid, followed by PCR screening of individual bacterial colonies using a primer specific to the plasmid vector and either the R-Loop (FIG. 16B) or the F-Loop (FIG. 16C) primer. Positive PCR products are then sent for direct sequencing using the vector-specific primer.
Reference is now made to FIG. 16B, which depicts a second method in which R-Stem primer and R-Loop primers are used in a nested-PCR approach. First, PCR is performed with the R-Stem primer and the primer that matches the 5′ adaptor sequence (5-ad primer). PCR products are then amplified in a second PCR using the R-Loop and 5-ad primers. As mentioned hereinabove, sequence data from the amplified hairpins is obtained in two ways. One way is the direct sequencing of the PCR products using the 5-ad primer. Another way is the cloning of the PCR products into a plasmid, followed by PCR screening of individual bacterial colonies using a primer specific to the plasmid vector and F-Stem primer. Positive PCR products are then sent for direct sequencing using the vector-specific primer. It should be noted that optionally an extended R-Loop primer is designed that includes a T7 sequence extension, as described hereinabove (FIG. 16A) for the R-Stem primer. This is important in the first sequencing option in cases where the PCR product is too short for sequencing.
Reference is now made to FIG. 16C, which depicts a third method, which is the exact reverse of the second method described hereinabove (FIG. 16B). F-Stem and F-Loop primers are used in a nested-PCR approach. First, PCR is performed with the F-Stem primer and the primer that matches the 3′ adaptor sequence (3-ad primer). PCR products are then amplified in a second PCR using the F-Loop and 3-ad primers. As in the other two methods, sequence data from the amplified hairpins is obtained in two ways. One way is the direct sequencing of the PCR products using the F-Loop primer. Another way is the cloning of the PCR products into a plasmid, followed by PCR screening of individual bacterial colonies using a primer specific to the plasmid vector and R-Stem primer. Positive PCR products are then sent for direct sequencing using the vector-specific primer. It should be noted that optionally an extended F-Loop primer is designed that includes a T3 sequence extension, as described hereinabove (FIG. 16A) for the F-Stem primer. This is important in the first sequencing option in cases where the PCR product is too short for sequencing and also in order to enable the use of T3 primer.
In an embodiment of the present invention, the three methods mentioned hereinabove may be employed to validate the expression of GAM FOLDED PRECURSOR RNA.
Reference is now made to FIG. 17A, which is a flow chart with a general description of the design of the microarray to identify expression of published miRNA oligonucleotides, and of novel GAM oligonucleotides of the present invention.
A microarray that identifies miRNA oligonucleotides is designed (FIG. 17B). The DNA microarray is prepared by Agilent according to their SurePrint Procedure (reference describing their technology can be obtained from the Agilent website, http://www.agilent.com). In this procedure, the oligonucleotide probes are synthesized on the glass surface. Other methods can also be used to prepare such microarray including the printing of pre-synthesized oligonucleotides on glass surface or using the photolithography method developed by Affymetrix (Lockhart D J et al., Nat Biotechnol. 14:1675-1680 (1996)). The 60-mer sequences from the design are synthesized on the DNA microarray. The oligonucleotides on the microarray, termed “probes” are of the exact sequence as the designed 60-mer sequences. Importantly, the 60-mer sequences and the probes are in the sense orientation with regards to the miRNA oligonucleotides. Next, a cDNA library is created from size-fractionated RNA, amplified, and converted back to RNA (FIG. 17C). The resulting RNA is termed “cRNA”. The conversion to RNA is done using a T7 RNA polymerase promoter found on the 3′ adaptor (FIG. 17C; T7 Ncol-RNA-DNA 3′Adaptor). Since the conversion to cRNA is done in the reverse direction compared to the orientation of the miRNA oligonucleotides, the cRNA is reverse complementary to the probes and is able to hybridize to it. This amplified RNA is hybridized with the microarray that identifies miRNA oligonucleotides, and the results are analyzed to indicate the relative level of miRNA oligonucleotides (and hairpins) that are present in the total RNA of the tissue (FIG. 18).
Reference is now made to FIG. 17B, which describes how the microarray to identify miRNA oligonucleotides is designed. miRNA oligonucleotide sequences or potential predicted miRNA oligonucleotides are generated by using known or predicted hairpins as input. Overlapping potential miRNA oligonucleotides are combined to form one larger sub-sequence within a hairpin.
To generate non-expressed sequences (tails), artificial sequences are generated that are 40 nts in length, which do not appear in the respective organism genome, do not have greater than 40% homology to sequences that appear in the genome, and with no 15-nucleotide window that has greater than 80% homology to sequences that appear in the genome.
To generate probe sequences, the most probable miRNA oligonucleotide sequences are placed at position 3 (from the 5′ end) of the probe. Then, a tail sub-sequence to the miRNA oligonucleotide sequence was attached such that the combined sequence length will meet the required probe length (60 nts for Agilent microarrays).
The tails method provides better specificity compared to the triplet method. In the triplet method, it cannot be ascertained that the design sequence, and not an uncontrolled window from the triplet probe sequence, was responsible for hybridizing to the probe. Further, the tails method allows the use of different lengths for the potential predicted miRNA oligonucleotide (of combined, overlapping miRNA oligonucleotides).
Hundreds of control probes were examined in order to ensure the specificity of the microarray. Negative controls contain probes which should have low intensity signal. For other control groups, the concentration of certain specific groups of interest in the library are monitored. Negative controls include tail sequences and non-hairpin sequences. Other controls include mRNA for coding genes, tRNA, and snoRNA.
For each probe that represents known or predicted miRNA oligonucleotides, additional mismatch probes were assigned in order to verify that the probe intensity is due to perfect match (or as close as possible to a perfect match) binding between the target miRNA oligonucleotide cRNA and its respective complementary sequence on the probe. Mismatches are generated by changing nucleotides in different positions on the probe with their respective complementary nucleotides (A <-> T, G<-> C, and vice versa). Mismatches in the tail region should not generate a significant change in the intensity of the probe signal, while mismatches in the miRNA oligonucleotide sequences should induce a drastic decrease in the probe intensity signal. Mismatches at various positions within the miRNA oligonucleotide sequence enable us to detect whether the binding of the probe is a result of perfect match or, alternatively, nearly perfect match binding.
Based on the above scheme, we designed a DNA microarray prepared by Agilent using their SurePrint technology. Table 11 is a detailed list of microarray chip probes
Known miRNA Oligonucleotides:
The miRNA oligonucleotides and their respective precursor sequences are taken from Sanger Database to yield a total of 186 distinct miRNA oligonucleotide and precursor pairs. The following different probes are constructed:
1. Single miRNA Oligonucleotide Probes:
From each precursor, 26-mer containing the miRNA oligonucleotide were taken, then assigned 3 probes for each extended miRNA oligonucleotide sequence: 1. the 26-mer are at the 5′ of the 60-mer probe, 2. the 26-mer are at the 3′ of the 60-mer probe, 3. the 26-mer are in the middle of the 60-mer probe. Two different 34-mer subsequences from the design tails are attached to the 26-mer to accomplish 60-mer probe. For a subset of 32 of Single miRNA oligonucleotide probes, six additional mismatches mutations probes were designed:
4 block mismatches at 5′ end of the miRNA oligonucleotide;
6 block mismatches at 3′ end of the miRNA oligonucleotide;
1 mismatch at position 10 of the miRNA oligonucleotide;
2 mismatches at positions 8 and 17 of the miRNA oligonucleotide;
3 mismatches at positions 6, 12 and 18 of the miRNA oligonucleotide; and
6 mismatches at different positions out of the miRNA oligonucleotide.
2. Duplex miRNA Oligonucleotide Probes:
From each precursor, a 30-mer containing the miRNA oligonucleotide was taken, then duplicated to obtain 60-mer probe. For a subset of 32 of probes, three additional mismatch mutation probes were designed:
2 mismatches on the first miRNA oligonucleotide;
2 mismatches on the second miRNA oligonucleotide; and
2 mismatches on each of the miRNA oligonucleotides.
3. Triplet miRNA Oligonucleotide Probes:
Following Krichevsky's work (Krichevsky et al., RNA 9:1274-1281 (2003)), head to tail ˜22-mer length miRNA oligonucleotide sequences were attached to obtain 60-mer probes containing up to three repeats of the same miRNA oligonucleotide sequence. For a subset of 32 probes, three additional mismatch mutation probes were designed:
2 mismatches on the first miRNA oligonucleotide;
2 mismatches on the second miRNA oligonucleotide; and
2 mismatches on each of the miRNA oligonucleotides.
4. Precursor with miRNA Oligonucleotide Probes:
For each precursor, 60-mer containing the miRNA oligonucleotide were taken.
5. Precursor without miRNA Oligonucleotide Probes:
For each precursor, a 60-mer containing no more then 16-mer of the miRNA oligonucleotide was taken. For a subset of 32 probes, additional mismatch probes containing four mismatches were designed.
Control Groups:
1. 100 60-mer sequences from representative ribosomal RNAs.
2. 85 60-mer sequences from representatives tRNAs.
3. 19 60-mer sequences from representative snoRNA.
4. 294 random 26-mer sequences from human genome not contained in published or predicted precursor sequences, placing them at the probe's 5′ and attached 34-mer tail described above.
5. Negative Control: 182 different 60-mer probes contained different combinations of 10 nt-long sequences, in which each 10 nt-long sequence is very rare in the human genome, and the 60-mer combination is extremely rare.
Predicted GAM RNAs:
There are 8642 pairs of predicted GAM RNA and their respective precursors. From each precursor, a 26-mer containing the GAM RNA was placed at the 5′ of the 60-mer probe and a 34-mer tail was attached to it. For each predicted probe, a mutation probes with 2 mismatches at positions 10 and 15 of the GAM RNA were added.
For a subset of 661 predicted precursors, up to 2 probes each containing one side of the precursor including any possible GAM RNA in it were added.
Microarray Analysis:
Based on known miRNA oligonucleotide probes, a preferred position of the miRNA oligonucleotide on the probe was evaluated, and hybridization conditions adjusted and the amount of cRNA to optimize microarray sensitivity and specificity ascertained. Negative controls are used to calculate background signal mean and standard deviation. Different probes of the same miRNA oligonucleotide are used to calculate signal standard deviation as a function of the signal.
For each probe, BG_Z_Score=(log(probe signal)−mean of log(negative control signal))/(log(negative control signal) standard deviation) were calculated.
For a probe with a reference probe with 2 mismatches on the miRNA oligonucleotide, MM_Z_Score MM_Z_Score=(log(perfect match signal)−log(reference mismatch signal))/(standard deviation of log(signals) as the reference mismatch log(signal)) were calculated.
BG_Z_Score and MM_Z_Score are used to decide whether the probe is on and its reliability.
Reference is now made to FIG. 17C, which is a flowchart describing how the cDNA library was prepared from RNA and amplified. The general procedure was performed as described previously (Elbashir S M, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 15:188-200) with several modifications, which will be described hereinbelow.
First, the starting material is prepared. Instead of starting with standard total RNA, the total RNA was size-fractionated using an YM-100 Microcon column (Millipore Corporation, Billerica, Mass., USA) in the present protocol. Further, the present protocol uses human tissue or cell lines instead of a Drosophila in vitro system as starting materials. Finally, 3 micrograms of size-fractionated total RNA was used for the ligation of adaptor sequences.
Libraries used for microarray hybridization are listed hereinbelow: “A” library is composed of a mix of libraries from Total HeLa YM100 RNA and Nuclear HeLa YM100 RNA; “B” library is composed of a mix of libraries from Total HEK293 YM100 RNA and Nuclear HEK293 YM100 RNA; “C” library is composed of a mix of YM100 RNA libraries from Total PC3, Nuclear PC3 and from PC3 cells in which Dicer expression was transiently silenced by Dicer specific siRNA; “D” library is prepared from YM100 RNA from Total Human Brain (Ambion Cat#7962); “E” library is prepared from YM100 RNA from Total Human Liver (Ambion Cat#7960); “F” library is prepared from YM100 RNA from Total Human Thymus (Ambion Cat#7964); “G” library is prepared from YM100 RNA from Total Human Testis (Ambion Cat#7972); and “H” library is prepared from YM100 RNA from Total Human Placenta (Ambion Cat#7950).
Library letters appended by a numeral “1” or “2” are digested by XbaI (NEB); Library letters affixed by a numeral “3” are digested by Xba1 and Spel (NEB); Library letters appended by a numeral “4” are digested by Xba1 and the transcribed cRNA is then size-fractionated by YM30, retaining the upper fraction consisting of 60 nts and longer; Library letters affixed by a numeral “5” are digested by Xba1 and the transcribed cRNA is then size-fractionated by YM30 retaining the flow-through fraction consequently concentrated with YM10 consisting of 30 nts-60 nts; Library letters affixed by a numeral “6” are digested by Xba1 and the DNA is fractionated on a 13% native acrylamide gel from 40-60 nt, electroeluted on a GeBaFlex Maxi column (GeBa Israel), and lyophilized; Library letters affixed by a numeral “7” are digested by Xba1 and the DNA is fractionated on a 13% native acrylamide gel from 80-160 nt, electroeluted and lyophilized.
Next, unique RNA-DNA hybrid adaptor sequences with a T7 promoter were designed. This step is also different than other protocols that create libraries for microarrays. Most protocols use complements to the polyA tails of mRNA with a T7 promoter to amplify only mRNA. However, in the present invention, adaptors are used to amplify all of the RNA within the size-fractionated starting material. The adaptor sequences are ligated to the size-fractionated RNA as described in FIG. 13, with subsequent gel-fractionation steps. The RNA is then converted to first strand cDNA using reverse transcription.
Next, the cDNA is amplified using PCR with adaptor-specific primers. At this point, there is the optional step of removing the tRNA, which is likely to be present because of its low molecular weight, but may add background noise in the present experiments. All tRNA contain the sequence ACC at their 3′ end, and the adaptor contains GGT at its 5′ end. This sequence together (GGTACC) is the target site for Ncol restriction digestion. Thus, adding the restriction enzyme Ncol either before or during PCR amplification will effectively prevent the exponential amplification of the cDNA sequences that are complements of the tRNAs.
The amplified DNA is restriction enzyme-digested with Xba1 (and, optionally, with Pst or Spel) to remove the majority of the adaptor sequences that were initially added to the RNA. Using the first set of RNA-DNA hybrid adaptors listed below, the first two sets of primers listed below, and Xba1 restriction digest yields the following cRNA products: 5′GGCCA-PRE/miRNA-UAUCUAG, where PRE is defined as GAM PRECURSOR (palindrome). Using the second set of RNA-DNA hybrid adaptors listed below, the second set of primers listed below, and Xba1 and Pst restriction digest yields the following, smaller cRNA products: 5′GG-PRE/miRNA-C*.
Then, cDNA is transcribed to cRNA utilizing an RNA polymerase e.g. T7 dictated by the promoter incorporated in the adaptor. cRNA may be labeled in the course of transcription with aminoallyl or fluorescent nucleotides such as Cy3- or Cy5-UTP and CTP among other labels, and cRNA sequences thus transcribed and labeled are hybridized with the microarray.
The following RNA-DNA hybrid adaptors are included in the present invention:
Name: T7 Ncol-RNA-DNA 3′Adapter
Sequence:
5′(5phos) rUrGrGCCTATAGTGAGTCGTATTA (3InvdT)3′
2. Name: 5Ada RNA-DNA XbaBseRI
Sequence: 5′ AAAGGAGGAGCTCTAGrArUrA 3′ or optionally:
3. Name: 5Ada MC RNA-DNA PstAtaBser
Sequence: 5′CCTAGGAGGAGGACGTCTGrCrArG 3′
4. Name: 3′Ada nT7 MC RNA-DNA
Sequence: 5′ (5phos) rCrCrUATAGTGAGTCGTATTATCT (3InvdT)3′
The following DNA primers are included in the present invention:
1. Name: T7 Ncol-RT-PCR primer
Sequence: 5′ TAATACGACTCACTATAGGCCA 3′
2. Name: T7Nhel Spel-RT-PCR primer
Sequence: 5′ GCTAGCACTAGTTAATACGACTCACTATAGGCCA 3′
3. Name: 5Ada XbaBseRI Fwd
Sequence: 5′ AAAGGAGGAGCTCTAGATA 3′
4. Name: Pst-5Ada XbaBseRI Fwd
Sequence: 5′ TGACCTGCAGAAAGGAGGAGCTCTAGATA 3′
or optionally:
5. Name: 5Ada MC PstAtaBser fwd
Sequence: 5′ ATCCTAGGAGGAGGACGTCTGCAG 3′
6. Name: RT nT7 MC Xbal
Sequence: 5′ GCTCTAGGATAATACGACTCACTATAGG 3′
Reference is now made to FIG. 18A, which demonstrates the detection of known miRNA oligonucleotides and of novel GAM oligonucleotides, using a microarray constructed and operative in accordance with a preferred embodiment of the present invention. Based on negative control probe intensity signals, we evaluated the background, non-specific, logarithmic intensity distribution, and extracted its mean, designated BG_mean, and standard deviation, designated BG_std. In order to normalize intensity signals between different microarray experiments, a Z score, which is a statistical measure that quantifies the distance (measured in standard deviations) that a data point is from the mean of a data set, was calculated for each probe with respect to the negative control using the following Z score formula: Z=(logarithm of probe signal BG_mean)/BG_std. We performed microarray experiments using RNA extracted from several different tissues and we calculated each probes maximum Z score. FIG. 18A shows the percentages of known, predicted and negative control groups that have a higher max Z score than a specified threshold as a function of max Z score threshold. The negative control group plot, included as a reference, considers probe with a max Z score greater then 4 as a reliable probe with meaningful signals. The sensitivity of our method was demonstrated by the detection of almost 80% of the known published miRNA oligonucleotides in at least one of the examined tissues. At a threshold of 4 for the max Z score, 28% of the predicted GAMs are present in at least one of the examined tissues.
Reference is now made to FIG. 18B, which is a line graph showing specificity of hybridization of a microarray constructed and operative in accordance with a preferred embodiment of the present invention and described hereinabove with reference to FIGS. 17A-17C.
The average signal of known miRNA oligonucleotides in Library A2 is presented on a logarithmic scale as a function of the following probe types under two different hybridization conditions: 50 C and 60 C: perfect match (PM), six mismatches on the tail (TAIL MM), one mismatch on the miRNA oligonucleotide (1 MM), two separate mismatches on the miRNA oligonucleotide (2 MM), three separate mismatches on the miRNA oligonucleotide (3 MM). The relative equality of perfect match probes and probes with the same miRNA oligonucleotide but many mismatches over the tail attest to the independence between the tail and the probe signal. At a hybridization temperature of 60 C, one mismatch in the middle of the miRNA oligonucleotide is enough to dramatically reduce the probe signal. Conducting chip hybridization at 60 C ensures that a probe has a very high specificity.
It is appreciated that these results demonstrate the specificity of the microarray of the present invention in detecting expression of miRNA oligonucleotides.
Reference is now made to FIG. 18C, which is a summary table demonstrating detection of known miRNA oligonucleotides using a microarray constructed and operative in accordance with a preferred embodiment of the present invention and described hereinabove with reference to FIGS. 17A-17C.
Labeled cRNA from HeLa cells and Human Liver, Brain, Thymus, Placenta, and Testes was used for 6 different hybridizations. The table contains the quantitative values obtained for each miRNA oligonucleotide probe. For each miRNA oligonucleotide, the highest value (or values) is given in bolded font while lower values are given in regular font size. Results for MIR-124A, MIR-9 and MIR-122A are exactly as expected from previous studies. The References column contains the relevant references in the published literature for each case. In addition to these miRNA oligonucleotides, the table shows other known miRNA oligonucleotides that are expressed in a tissue-specific manner. The results indicate that MIR-128A, MIR-129 and MIR-128B are highly enriched in Brain; MIR-194, MIR-148 and MIR-192 are highly enriched in Liver; mIR-96, MIR-150, MIR-205, MIR-182 and MIR-183 are highly enriched in Thymus; MIR-204, MIR-10B, MIR-154 and MIR134 are highly enriched in Testes; and MIR-122, MIR-210, MIR-221, MIR-141, MIR-23A, MIR-200C and MIR-136 are highly enriched in Placenta. In most cases, low but significant levels are observed in the other tissues. However, in some cases, miRNA oligonucleotides are also expressed at relative high levels in an additional tissue.
It is appreciated that these results reproduce previously published studies of expression of known miRNA oligonucleotides. These results demonstrate the reliability of the microarray of the present invention in detecting expression of published miRNA oligonucleotides, and of novel GAM oligonucleotides of the present invention.

DETAILED DESCRIPTION OF TABLES

Table 1 comprises data relating the SEQ ID NO of oligonucleotides of the present invention to their corresponding GAM NAME, and contains the following fields: GAM SEQ-ID: GAM SEQ ID NO, as in the Sequence Listing; GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); GAM RNA SEQUENCE: Sequence (5′ to 3′) of the mature, “diced” GAM RNA; GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; GAM POS: Dicer-cut location (see below); and
Table 2 comprises detailed textual description according to the description of FIG. 1 of each of a plurality of novel GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; PRECUR SEQ-ID:GAM precursor Seq-ID, as in the Sequence Listing; PRECURSOR SEQUENCE: Sequence (5′ to 3′) of the GAM precursor; GAM DESCRIPTION: Detailed description of GAM oligonucleotide with reference to FIG. 1; and
Table 3 comprises data relating to the source and location of novel GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); PRECUR SEQ-ID: GAM precursor SEQ ID NO, as in the Sequence Listing; GAM ORGANISM: identity of the organism encodes the GAM oligonucleotide; SOURCE: For human GAM—chromosome encoding the human GAM oligonucleotide, otherwise—accession ID (GenBank, NCBI); STRAND: Orientation of the strand, “+” for the plus strand, “−” for the minus strand; SRC-START OFFSET: Start offset of GAM precursor sequence relative to the SOURCE; SRC-END OFFSET: End offset of GAM precursor sequence relative to the SOURCE; and
Table 4 comprises data relating to GAM precursors of novel GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); PRECUR SEQ-ID: GAM precursor Seq-ID, as in the Sequence Listing; GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; PRECURSOR-SEQUENCE: GAM precursor nucleotide sequence (5′ to 3′); GAM FOLDED PRECURSOR RNA: Schematic representation of the GAM folded precursor, beginning 5′ end (beginning of upper row) to 3′ end (beginning of lower row), where the hairpin loop is positioned at the right part of the draw; and
Table 5 comprises data relating to GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; GAM RNA SEQUENCE: Sequence (5′ to 3′) of the mature, “diced” GAM RNA; PRECUR SEQ-ID: GAM precursor Seq-ID, as in the Sequence Listing; GAM POS: Dicer-cut location (see below); and
Table 6 comprises data relating SEQ ID NO of the GAM target gene binding site sequence to TARGET gene name and target binding site sequence, and contains the following fields: TARGET BINDING SITE SEQ-ID: Target binding site SEQ ID NO, as in the Sequence Listing; TARGET ORGANISM: identity of organism encode the TARGET gene; TARGET: GAM target gene name; TARGET BINDING SITE SEQUENCE: Nucleotide sequence (5′ to 3′) of the target binding site; and
Table 7 comprises data relating to target-genes and binding sites of GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; GAM RNA SEQUENCE: Sequence (5′ to 3′) of the mature, “diced” GAM RNA; TARGET: GAM target gene name; TARGET REF-ID: For human target genes—Target accession number (RefSeq, GenBank); Otherwise—the location of the target gene on the genome annotation. TARGET ORGANISM: identity of organism encode the TARGET gene; UTR: Untranslated region of binding site/s (3′ or 5′); TARGET BS-SEQ: Nucleotide sequence (5′ to 3′) of the target binding site; BINDING SITE-DRAW: Schematic representation of the binding site, upper row represent 5′ to 3′ sequence of the TARGET, Lower row represent 3′ to 5′ Sequence of the GAM RNA; GAM POS: Dicer-cut location (see below); and
Table 8 comprises data relating to functions and utilities of novel GAM oligonucleotides of the present invention, and contains the following fields: GAM NAME: Rosetta Genomics Ltd. nomenclature (see below); GAM RNA SEQUENCE: Sequence (5′ to 3′) of the mature, “diced” GAM RNA; GAM ORGANISM: identity of the organism encoding the GAM oligonucleotide; TARGET: GAM target gene name; TARGET ORGANISM: identity of organism encode the TARGET gene; GAM FUNCTION: Description of the GAM functions and utilities; GAM POS: Dicer-cut location (see below); and
Table 9 comprises references of GAMs target genes and contains the following fields: TARGET: Target gene name; TARGET ORGANISM: identity of organism encode the TARGET gene; REFERENCES: reference relating to the target gene; and
Table 10 comprises data relating to novel GR (Genomic Record) polynucleotides of the present invention, and contains the following fields: GR NAME: Rosetta Genomics Ltd. nomenclature (see below); GR ORGANISM: identity of the organism encoding the GR polynucleotide; GR DESCRIPTION: Detailed description of a GR polynucleotide, with reference to FIG. 9; and
Table 11 comprises data of all sequences printed on the microarray of the microarray experiment, as described herein above with reference to FIG. 17 and include the following fields: PROBE SEQUENCE: the sequence that was printed on the chip PROBE TYPE: as described in detail in FIG. 17 in chip design section and summarized as follows: Known: published miRNA sequence; Known_mis1: similar to published miRNA sequence, but with 1 mismatch mutation on the miRNA sequence; Known_mis2: similar to published miRNA sequence, but with 2 mismatch mutations on the miRNA sequence; Known_mis3: similar to published miRNA sequence, but with 3 mismatch mutations on the miRNA sequence; Known_mis4: similar to published miRNA sequence, but with 6 mismatch mutations on regions other than the miRNA sequence; Predicted: predicted GAM RNA sequences; Mismatch: sequences that are similar to predicted GAM RNA sequences but with 2 mismatches; Edges 1: left half of GAM RNA sequences; Edges2: right half of GAM RNA sequences extended with its hairpin precursor (palindrome); Control1: negative control; Control2: random sequences; Control3: tRNA; Control4: snoRNA; Control5: mRNA; Control6: other; GAM RNA SEQ ID/MIR NAME: GAM oligonucleotide using Rosetta Genomics Ltd. Nomenclature (see below) or published miRNA oligonucleotide terminology; GAM RNA SEQUENCE: Sequence (5′ to 3′) of the mature, “diced” GAM RNA; LIBRARY: the library name as defined in FIG. 17C; SIGNAL: Raw signal data for library; BACKGROUND Z-SCORE: Z-score of probe signal with respect to background, negative control signals; MISMATCH Z-SCORE: Z-score of probe signal with respect to its mismatch probe signal; and
Table 12 comprises data related to the GAM RNA SEQUENCEs included in the present invention that were validated by laboratory means. If the validated sequence appeared in more than one GAM precursor, the GAM RNA SEQ-ID indicated may be arbitrarily chosen. The table includes the following fields: VALIDATION METHOD: the type of validation performed on the sequence. The microarray validations are divided into four groups: a) “Chip strong” refers to GAM oligonucleotide sequences whose intensity (SIGNAL) on the microarray “chip” was more than 6 standard deviations above the background intensity, and the differential to the corresponding mismatch intensity was more than 2 standard deviations, where in this case the standard deviation is of the intensity of identical probes; b) “Chip” refers to GAM oligonucleotide sequences, whose intensity was more than 4 standard deviations above the background intensity; c) “Sequenced” refers to GAM oligonucleotide sequences that were sequenced; and d) “Chip strong, Sequenced” refers to miRNA oligonucleotide sequences that were both detected in the microarray as “Chip strong” and sequenced. “Sequenced” is described hereinabove with reference to FIG. 13. Other validations are from microarray experiments as described hereinabove with reference to FIGS. 17A-C and 18A-C; SIGNAL: a raw signal data; BACKGROUND Z-SCORE: a Z-score of probe signal with respect to background, negative control signals; MISMATCH Z-SCORE: a Z-score of probe signal with respect to its mismatch probe signal; and
Table 13 comprises sequence data of GAMs associated with different bacterial infections. Each row refers to a specific bacterial infection, and lists the SEQ ID NOs of GAMs that target genes associated with that bacterial infection. The table contains the following fields: ROW#: index of the row number; INFECTION NAME: name of the infecting organism; and SEQ ID NOs OF GAMS ASSOCIATED WITH INFECTION: list of sequence listing IDs of GAMs targeting genes that are associated with the specified infection.
The following conventions and abbreviations are used in the tables: The nucleotide “U” is represented as “T” in the tables, and;
GAM NAME or GR NAME are names for nucleotide sequences of the present invention given by Rosetta Genomics Ltd. nomenclature method. All GAMs/GRs are designated by GAMx/GRx where x is a unique ID.
GAM POS is a position of the GAM RNA on the GAM PRECURSOR RNA sequence. This position is the Dicer-cut location: A indicates a probable Dicer-cut location; B indicates an alternative Dicer-cut location.
All human nucleotide sequences of the present invention as well as their chromosomal location and strand orientation are derived from sequence records of UCSC-hg16 version, which is based on NCBI, Build34 database (April, 2003).

All bacterial sequences of the present invention as well as their genomic location are derived from NCBI, RefSeq database.



VALIDATION	BACKGROUND	MISMATCH	GAM RNA

GAM RNA SEQUENCE	METHOD	SIGNAL	Z-SCORE	Z-SCORE	SEQ-ID

ACTCACTGCAACCTCCACCTCC	Sequenced			323

ACTGCACTCCAGCCTGGGCTAC	Sequenced			13

AATCACTTGAACCCAAGAAGTG	Sequenced			5

AATCGCTTGAACCCAGGAAGTG	Sequenced			6

TTCAAGTGTTTAAGTTCTGCTT	Sequenced			305

AGGCAGAGAGGACCAGAGACT	Sequenced			331

CACTGCACTCCAGCCCGAGCAA	Sequenced			46

CCCGGGTGGAGCCTGGGCTGTG	Sequenced			361

GGGCGTGGAGCTGGAATGATGT	Sequenced			125

TGATAGATCCATATTTTGGTAA	Sequenced			279

AGCAAGACCAGGGTTTTGTGTT	Sequenced			326

TCACTGCAACCTCCACCTCCCA	Sequenced			198

ATTGTTGCCCATGTTTTTATTT	Sequenced			40

CTGGACTGAGCTCCTTGAGGCC	Sequenced			383

AGGCCAAGAAGGAAGCAGAGG	Sequenced			25

ATTAGGAGAGTGGGTGCTAAGT	Sequenced			38

AGTTTGTGTAAGAAAAGC	Sequenced			338

AGGAAAAAAATTAATGTGAGTC	Sequenced			22

TCACTGCAACCTCCACCAGCCT	Sequenced			197

GTGACAGTGAATCTAGACAGAC	Sequenced			134

TATTCATTGCCCATGTTTGTGA	Sequenced			262

TGGGTTTTGTTTGTACAGTGTA	Sequenced			229

CTCAGCTCATCCACTAAATCCC	Sequenced			377

TCACTGCAACCTCCACCTTCAG	Sequenced			263

GGGAAATAATTAATGTGAAGTC	Sequenced			124

TGGAGGAGAGTTTGTCAGTATAG	Sequenced			298

GGAATGGTGGTTGTATGGTTG	Sequenced			116

TCACTGCAACCTCCACCTTCCG	Sequenced			201

TTCTGATGGTTAAGTTCTGTCA	Sequenced			306

AGGGCAGGAGGTCCGTCCCTTC	Sequenced			27

TCACTGCAACCTCCACCACGTG	Sequenced			196

TCTAAGAGAAAGGAAGTTCAGA	Sequenced			272

GAAGTTTGAAGCCTGTTGTTCA	Sequenced			95

CTAGACTGAAGCTCCTTGAGGA	Sequenced			74

AATTGCTTGAACCCAGGAAGTGGA	Sequenced			8

CACTGCAACCTCCACCTCCTGG	Chip strong,	31393	19.150194	22.611071	45
	Sequenced

TCACTGCAACCTCCACCTCCCG	Chip strong,	31810	20.186802	16.772465	199
	Sequenced

TCACTGCAACCTCCACCTCCTG	Chip strong,	45662	20.504339	18.911047	200
	Sequenced

ATGGTAGCTGTCCACATCAGGA	Chip strong	8208	25.85717	21.352978	36

TCAGCTCCTACCCCGGCCCCAG	Chip strong	8279.5	11.228731	17.399603	204

GTTTCTCTGGGCTTGGCAT	Chip strong	8298	10.689093	5.6611276	257

TGGTCTGGCCCACATGGTC	Chip strong	8349	13.022524	4.8629713	231

GTGCTGGTGCTCGCTCCTCTGG	Chip strong	8165	11.725875	9.7062302	251

CTCAGGTGATCCACCCCTCTTG	Chip strong	8190	8.7424583	3.9819176	75

TGCAGGTTGCTGGTCTGATCTC	Chip strong	8079	24.743416	17.869699	283

AGTCATTATCTCCTGGACC	Chip strong	7790	10.371323	17.396904	30

GCTGCACCCCAGCCTGGGTAAC	Chip strong	7858	6.2366548	20.271864	162

CACTTCCCTTCTCTGCTCATGG	Chip strong	7886.5	8.1030474	7.7415953	347

TGCTGGCTATCCTGCGCCTTTC	Chip strong	7903	10.469044	13.746831	225

GGCTGCTGGTTTCTTGTTTTAG	Chip strong	7926	12.94939	11.212504	176

CTTCCTGCCTCTCGCCGCCCGC	Chip strong	7982	10.846725	2.7860351	89

CTGCTCTGGTTTCCTCTGTC	Chip strong	7506.5	7.7015729	15.622507	86

GCCTCCAGGTCGGTCTTTCTCT	Chip strong	7529	13.077046	6.7496343	104

CCCTCTTGGCTTCTATCCCACC	Chip strong	7596	7.1978688	6.3785648	363

CAGCTGGTGCTTGCCTGGCTAA	Chip strong	7373	13.676201	7.9258513	351

TCTCCCAGATCCTTTAGCCTCC	Chip strong	7384.5	14.663905	2.166656	274

TTTCTTGGGCCGTGTGCTGGT	Chip strong	7386	8.0159159	10.662634	248

ATCACTTTGAGTCCAGGAGTTT	Chip strong	7335	6.5335536	19.718058	32

GAGCCGCCCTCCACGATGTCCC	Chip strong	7252	8.6663809	14.735928	142

CCTCACTCAGGTTTGGACCCTG	Chip strong	7301	15.895414	5.3846102	61

GGGTTACTCTGTGTTGGTCAGG	Chip strong	7310	8.6937799	12.815997	129

TGCTCTGATTTTTGCCCCAGC	Chip strong	7060.5	10.413313	7.7476549	291

GCTGTTTTCCCATAGCTGGTCA	Chip strong	7061	19.803032	6.222959	164

GCTAGGCTGCTGGCCACTGAGG	Chip strong	6972.5	13.127683	19.686853	159

TGCTTGCTGTGGTTGGCTGGTA	Chip strong	6974	21.75724	11.332961	296

TCAGCCTCCTCCACCCCAGAGT	Chip strong	6996.5	14.03341	7.0927162	264

GGGGAACGCGCTGGCCCGCGCC	Chip strong	7005	6.2445078	11.806351	127

CTCTGTGATATGGTTTGTAATA	Chip strong	6862	19.265455	13.692534	84

CATTCTGTGAGCTGCTGGCTTT	Chip strong	6884	11.220102	9.6062307	52

CTCGACTTCCCTGGCTTGCGTGA	Chip strong	6890	6.5380254	11.584653	78

GGCGGCCCAGGCGCTTGGAGAT	Chip strong	6899.5	8.1672001	10.434432	172

TGCCGCCCGGCCATCTCGGCTC	Chip strong	6915.5	13.391404	5.9536037	220

TCTCTATGCCATGCTGGCCT	Chip strong	6926	17.665062	2.5852687	217

ACATTCTCTGATTGGTGCCTCC	Chip strong	6695	12.723179	6.4453721	319

CTGTGCTCTTTCCACGGCCCCA	Chip strong	6477.5	13.662484	9.3280506	139

AAGGCCGCCCCTTCATGCTCCT	Chip strong	6358.5	9.1175785	8.5895061	1

CAGCAGCTCAGCCTCCTTCCCA	Chip strong	6588	11.002058	9.0820408	349

CAGTTTGTCCCCATGGCCATGT	Chip strong	6591.5	13.401958	5.2375259	354

TGGAGCTGGGTCTGGGGCA	Chip strong	6426	15.46969	17.843594	297

CCTGGTCGGCGTGGTGACGGCG	Chip strong	6434.5	6.2044091	6.2762375	369

TCCTACGGTGGCCACAGTCTGG	Chip strong	6256	7.9984035	3.2358623	210

CGTTCACTCCCTTGCCCCTCGG	Chip strong	6280.5	7.0008011	9.7373304	73

TGTCTGGCTTTCTTCAGTTAGC	Chip strong	6191	9.9906111	15.989508	236

TGCTGCACCCTCTGCCTCCGGG	Chip strong	6094.5	6.9428978	10.588869	293

GCAGCATCCCGGCCTCCACTGT	Chip strong	5995	7.2606683	11.881517	147

TGTGGTAGTCACGGCCCGCCAC	Chip strong	5909.5	23.027369	15.816967	304

CTTGCCTGCCCTGTGTCATAAA	Chip strong	5903.5	13.361271	3.0393276	91

TTCACTGCTCTAGCCCTAATTT	Chip strong	5739	15.599205	7.8376389	240

TCCATTGGCCTTTTATCCTAGA	Chip strong	5760	15.329782	8.1126537	209

TGCCTAGCCAAGTCCAGTATTT	Chip strong	5823	17.976177	16.478537	221

TTCTGGCTTCTCCCAGGCGGCC	Chip strong	5582	8.2352791	10.879703	243

ATGGCCCTCTTATCACAGCTCC	Chip strong	5586.5	21.480997	6.3762493	342

ACTGCACTCCATCCAGCCTGGC	Chip strong	5668	7.6480083	10.938603	324

TGCCTGCCCCAGCTGAGATATC	Chip strong	5686	10.380668	15.221783	287

GCTCGCTGGGGTCTGCAGGCGG	Chip strong	5502	7.7859778	10.874097	111

GCAGCTCCTGGAGGTGAGAGGCG	Chip strong	5368	7.8018293	15.956004	100

CTCATTGTAGCCTCCAGTTCTTG	Chip strong	5375	10.634505	9.6296253	379

AGGCTGGTTAGATTTGTGGTCT	Chip strong	5392	20.112637	16.324888	26

GCTGCACTTCAGCCTGGGTGTC	Chip strong	5310	7.5533419	15.940791	113

GCCCTTTGTGTCTGGCTGGGGT	Chip strong	5320	11.978069	10.261797	152

TTCTCTGTGCTGGGTCCTGAGG	Chip strong	5272.5	8.1261625	9.2259359	242

AGATTTCCCTTCCTGCTTGCCT	Chip strong	5251	6.0291886	13.065763	17

TGCGTTCCAGTTGCTGCCAGGC	Chip strong	5079	11.194171	5.7294831	290

CTGGCTAAGATCCAAGAAAGGC	Chip strong	5036	14.178236	6.6532001	385

TCATTGCAACCTCCTCCTGGGT	Chip strong	5039.5	18.95397	9.7537737	207

TTGACATGCCTCCTACATGATC	Chip strong	5065	12.953059	10.809283	307

CCTGCTCTCTGTTCTTAAGCTT	Chip strong	5021	9.0648565	7.4354005	64

TGCACCACTGCACCCCAGTCTG	Chip strong	5009	7.3463378	16.848854	281

TGCTGCCCTAAGACCACCTT	Chip strong	4950	11.124713	13.249466	294

GGGAGTTGTGGTTGGCTTCTGG	Chip strong	4978	8.3206406	9.2158394	179

GGCCGTGGTCGCTGACTCTCGT	Chip strong	4980	6.9448657	12.094063	120

TAGGTATGGCTTGTGGCACAGC	Chip strong	4840	23.281979	15.36544	261

GCGCCGCCATCCGCATCCTCGT	Chip strong	4801	16.34218	9.281786	107

CTGGTGTTGGGTCTTGCTTTTA	Chip strong	4756	6.5764294	8.8639517	138

ATGGGCCTCCTATTATCCCCAT	Chip strong	4745.5	13.363207	5.1394033	34

CGCCCAGGCTGGAGTGCCAGTG	Chip strong	4722	9.6376123	13.758563	69

CGACCTTGTGATCCTCCCGCCT	Chip strong	4594	7.4134154	4.4487605	370

CTCAGTGCAACCTCCGCCTACT	Chip strong	4516	8.8905106	13.512998	76

GGCTCTGGCTTTGGAGGAGCAG	Chip strong	4483.5	6.8781896	14.473881	174

GGGCTTTTGGAATGGTCTGT	Chip strong	4463	9.6709318	2.0551727	126

AGTCGCTGGACCATCAGAGCCT	Chip strong	4419	12.240126	13.100382	335

GGTGGTGGAGCGGGCCCAGGCC	Chip strong	4320.5	7.4591732	12.328825	185

TCCAGCTGTCCACGTCTTCCTG	Chip strong	4070	6.5770264	7.9605851	265

ATGGTACTCCAGCCTGGGTGAC	Chip strong	4173	7.3957338	16.409479	35

ATTCTGTGCTAACTGCAGGCCA	Chip strong	4140	19.305922	11.530575	343

GACCTCGTGATCCGCCTGCTTT	Chip strong	4080.5	7.6009617	13.947659	97

TGGTGCAGCGTGTGGTGGCTCT	Chip strong	4082.5	9.6208868	12.887189	302

TGGTCGGGCTGCATCTTCCGGC	Chip strong	4093	8.0100813	2.1106353	230

CACTGCAGCCTCCATCTCTGGG	Chip strong	4050	6.9180322	10.574921	47

ATGGTGCTGGTGGGAGTGTATT	Chip strong	4053	18.971554	14.625937	37

TGCCTGCCGTTAAATGTTACTT	Chip strong	3936	12.749383	11.509386	222

GACCTTGTGATCCGCCCACTTT	Chip strong	3834	7.5950313	9.0545225	141

CAACTCACTGCGGCCTCAACCT	Chip strong	3783	9.680912	5.8278494	41

CTGGAGGAGCTGCCATG	Chip strong	3669	12.842446	14.933422	384

TAGCTCCTCCCAGATCTCATCT	Chip strong	3659	10.385338	3.9473054	192

TTGGGGGAGGCCTGCTGCCCAT	Chip strong	3549	9.3567915	8.3044834	310

GTTGGTCTTCATTAAATGCTTT	Chip strong	3499.5	17.153486	5.8892236	255

GGTGGCTATGGCTGTGCTCGC	Chip strong	3426.5	15.917648	2.9563422	132

GATGTCGTGATCCACCCGCCTT	Chip strong	3425	7.313684	10.200798	145

AGTGGCGTGATCTCGGCTCGGT	Chip strong	3395	8.8775339	14.742507	336

GTGCTTAAAGAATGGCTGTCCG	Chip strong	3362	26.398634	13.195816	252

TCACTGCAAGCTCCACCCTCCG	Chip strong	3370	12.960393	9.7885542	202

CGGCACTGTAGTCTGGCTGGGA	Chip strong	3297	6.7212648	9.1534166	374

CTGGCTAGATGTGTGGCCATGA	Chip strong	3221	21.032122	14.058989	137

AACCTTGTGATCCACCCACCTT	Chip strong	3034	7.7903786	12.639959	313

AGCTGGCTTACTTGAGATGCAT	Chip strong	3049	8.8567095	7.4132333	329

GGGGCTTCTAGGGTGCCAGATC	Chip strong	3012.5	13.356146	7.901947	181

GGCCCAGGTTGGAGTGCAGTGA	Chip strong	2994	8.0930119	10.374014	168

GGCCCAGTGCAAGCTCTTTCTG	Chip strong	2960	7.6298795	6.4523926	118

GCCCTTGAAGCTCTGACCCGCT	Chip strong	2947	7.6962008	2.815666	151

GCTGGCTCCACCTGCTGCCAGG	Chip strong	2916	6.3332305	13.052609	115

CCACTGAGGTAGCTGGTGACTG	Chip strong	2861	16.719574	7.8953633	54

CCTCCGGTCATTGTGCGGGCCT	Chip strong	2835	12.644177	5.132216	366

AGGATCTTGCTATGTTGGCCAG	Chip strong	2784	10.949057	7.9714575	330

TGGTGCTAGTTAAATCTTCAGG	Chip strong	2715	17.999035	10.341267	232

GCACTGCTGCCTCCTGG	Chip strong	2627	6.3458524	7.414557	99

TTATAATGTATAGCTGTGCCTG	Chip strong	2566.5	15.056374	8.2182913	238

TGCTTCTAGGGAGGCCGCAGGA	Chip strong	2554	12.58359	11.930317	295

TAGAACTATGGCTATGTGCCA	Chip strong	2523.5	18.843672	7.4688845	259

GACCCATCCTCCACTTGGCAGC	Chip strong	2498	6.505065	6.8388047	96

GCCTAGTGGATTTGAAGGGCC	Chip strong	2352	20.613605	8.8114462	153

TGCCCACTGCTGGCCACCACCC	Chip strong	32112	15.630626	16.785101	219

GGCTGGCCCCATCCAGGCTGGCA	Chip strong	65518	10.117671	10.864906	121

ACAAAGCGCTTCTCTTTAGAGT	Chip strong	65518	11.238881	26.766436	9

GGGGCTGGTCTTTCCACTTACT	Chip strong	65518	11.24554	19.391401	180

GGAGGCTGGCCTTCAGACGGGT	Chip strong	65518	12.034198	25.266558	166

ACGCGCTGGGGCGCTGGCCAAT	Chip strong	65518	13.337035	9.5484018	12

ACAAAGTGCCTCCTTTTAGAGT	Chip strong	65518	13.412503	32.421429	10

CGCCTGGCCCCCAGTACTTTGT	Chip strong	65518	14.386203	22.674049	373

GCCTGGCCTAAATTAGTAATTT	Chip strong	65518	14.47023	33.939186	155

CCCTCTGGCCCCTGTGGTGGAT	Chip strong	65518	14.648276	19.804953	362

CTGCCTGCCTGGCCCAGGAACC	Chip strong	65518	14.752467	36.164337	381

CGCCCGCTGGCCCTGCGATCTC	Chip strong	65518	15.196337	33.776985	70

AGGACCTGTCCCCTGGCCCACT	Chip strong	65518	15.796532	15.770715	24

CAGCAGCACACTGTGGTTTGTA	Chip strong	65518	16.623587	30.172779	348

CCGCCTGGCCCATTGCAGGGCA	Chip strong	65518	19.692606	29.045151	365

CACTGCACTCCAGCTCTGGGT	Chip strong	65518	20.15584	31.571056	345

ACAAAGTGCCTCCCTTTAGAGT	Chip strong	65518	22.461653	34.028076	316

CCCCACTGTCCCCGGAGCTGGC	Chip strong	65518	22.799175	24.102064	358

GGCGCTGGCCTGTGGGATCCCG	Chip strong	65518	24.841112	31.449797	171

GCGGCGGCGGTAGCAAAAATGA	Chip strong	65518	27.5298	22.089998	109

AGGGTTGTGTGCTGGCCGCTGG	Chip strong	65518	29.01285	32.102142	28

GGTGGCCCCTGGGAGATGCTGG	Chip strong	65518	31.295538	14.111359	131

CACTGCACTCCAGCCTGGGCAA	Chip strong	65518	36.446095	33.140068	12852

TGTGCTGGCCTTTGGTGACTTC	Chip strong	65518	44.612064	26.016636	237

CATGCTGGCCCACACCCGCTGC	Chip strong	57891	37.069935	17.358248	50

GGCTTCCTGCCTCGGGCTGGCC	Chip strong	58372	13.006404	4.4936109	177

GCCTGGCCTAATTCCAGCATTT	Chip strong	62842.5	16.076189	31.293688	156

GAAGGGGGAAGAGAGCTGGCCG	Chip strong	63993	20.677708	18.040138	94

CCCGGCACCTCCGCTGCACAC	Chip strong	50589.5	17.716768	10.848449	360

ATGCCACTGCGCTCCAGCCTGA	Chip strong	50941.5	15.106459	30.447573	341

CCCCACTGTTTTCTTCATCCTA	Chip strong	50957	32.576454	4.8442335	359

CTTGGAGTAGGTCATTGGGTGG	Chip strong	51071	16.39068	33.942337	92

AGGTGCTGGGGCTTGGCCTGCT	Chip strong	54992	14.781937	19.839622	333

TGCCCGGATACCCCTGGCCTC	Chip strong	46111	13.316625	10.030684	285

ATTGCACTCCAGCCTGAGCAAA	Chip strong	46579	22.505102	33.557095	39

TCTCTTCGCTGGCCCTCGGGGA	Chip strong	47791.5	15.379544	20.008915	276

CCGTCCCCGGTGCTGCCTGCGC	Chip strong	48514	9.4747534	7.9190497	60

TGCTAGCTGCCCGAAGGTCTCA	Chip strong	39989	47.058292	15.67876	223

CCTGGCCGCTGTGCCCCCT	Chip strong	40002	11.873036	10.703612	65

ACACTTTGCCCCTGGCCGCCTT	Chip strong	42189	12.009233	22.436626	317

TGACCTCCTTTCTCGACTAATT	Chip strong	43651	10.281033	24.914602	278

CTGCTGCGCTGGCCGTCACGGT	Chip strong	45168	18.758972	18.507338	382

TTATTGCACTCCAGCCTGGGTA	Chip strong	45303	21.338472	22.149384	239

CTCAGTGCTGCTGGCTCCTGTC	Chip strong	30057	40.88406	25.543219	378

GACCCCTAAACCCGCTGGGCTG	Chip strong	30088.5	13.552105	6.4749699	140

CCTGGCTCTGGCTTCCTGTTGT	Chip strong	34525	11.373339	6.4300051	368

ACCCTGGCCGACTGCCCCTT	Chip strong	35652	12.982363	11.41268	11

GCCTGGCCTCCTACAGTACTTT	Chip strong	35866	15.014146	23.263319	157

GCCCTTCGGAAAGCGTCGCCTG	Chip strong	37481	13.375318	6.6135831	150

TGCCTGGCCTCCTGATTCCCTC	Chip strong	37634.5	13.004288	2.9085336	288

CCAGACCATTTTGCCTTACC	Chip strong	38076	30.955603	11.095823	55

CGTAAGTCACAGCGCCTGGCCC	Chip strong	38826	11.506068	25.787857	72

CAGGCTCTTCCCTCTGGCCAAG	Chip strong	25089	10.865691	11.601097	352

GATGAGTTTGCCTGGCCTGCAG	Chip strong	25445.5	12.297516	17.035336	143

GCTGTAAGTCACCTGGCCCGAT	Chip strong	26191	8.8471966	25.053482	163

AGAAGGGCTGGCAGGAGTT	Chip strong	26652	14.563484	25.132761	16

TGCCTGGCCTCTTCAGCACTTC	Chip strong	27021	10.873885	26.68429	289

GGTGCCCCATCGCGGGTGGCTG	Chip strong	27077	14.316696	22.61035	130

GCTCCTGGCCGGGCTGCTCCTG	Chip strong	27106	14.495318	9.280777	161

AAGTGCTCATAGTGCAGGTAGT	Chip strong	27166.5	9.1624584	28.31859	4

CAGGAAAAGGCGGCTCGGGGCT	Chip strong	27684.5	9.7338009	6.1309323	49

TCACGCGCCCTCCTGGGCCCTG	Chip strong	28630	10.411592	10.865385	195

GGCGTGCCCTGGCCCCGAGGCT	Chip strong	28813	10.987214	21.873014	173

TCCTGGGGCTTGTCGCTGGCCA	Chip strong	28926	12.960393	7.4913173	216

GCTTCAGAGAGGGGTGAAGCTG	Chip strong	21900	17.158428	13.963737	165

CTCTCCTTGGCCACCTCCATGA	Chip strong	23276	12.960393	7.0737572	81

GGCTGGTGGCTGGTTCTGGACC	Chip strong	20736.5	31.680035	17.914019	122

CACCCGCTGGTCCCTGCAGTTC	Chip strong	20816	8.5344362	27.261486	42

CCCTGGCTCACTTTCTGTTGTG	Chip strong	20839	26.185976	5.4283981	364

GGTAGTCTTTGTCCCCTGGC	Chip strong	20872	12.44091	3.1238594	182

CATCACCCCCAGACCTCAGTGC	Chip strong	20958.5	35.708847	4.6072259	355

GGCTGGTTAGATTTGTGGTCTT	Chip strong	21258	33.569485	15.757149	123

TTGGTCCCCTTCAACCAGCTAC	Chip strong	20228	9.5504265	23.87529	246

TCAGGGGTTGGCTTGTTGTGTT	Chip strong	20519.5	8.8405285	21.048086	206

TACTGCACTCCAGCCTTGCCAA	Chip strong	18364	10.029301	16.731598	258

AATTGCACGGTATCCATCTGTA	Chip strong	18407	8.3120737	26.950815	7

TGGTTCTTCGCTGGGCGGCTGC	Chip strong	18451	17.683105	11.562138	234

CCCTGCCTGTCCTGGTCCCGTT	Chip strong	18466	9.747386	21.814604	59

TCTCCACAGCTGGCCCCCAAGA	Chip strong	19483.5	23.591568	26.742323	273

CCTCGCTCTCCATTCGGCCCTC	Chip strong	9378.5	6.9943829	8.7534571	367

GGCCGGGTGCTCTGGAGGTGCT	Chip strong	14393	11.734104	12.172738	119

AGCTCCTGGCTTCAAGCAATCC	Chip strong	14107	10.339123	18.669428	20

TTTAAATCACAACTCTGCCCCT	Chip strong	15129	15.825633	8.2785378	247

GTAGCTGTGTTCATTCTGGATG	Chip strong	15186.5	37.683685	11.412519	187

AAGTGCTAGTGAGTCTATTGTA	Chip strong	15263	30.581371	17.914198	3

GCCCCAGCTCACCGGCTCACTG	Chip strong	15345	20.667051	7.4258513	103

GTGCGGCCTGGCCTTCAAGTGG	Chip strong	15350	9.6908836	19.487803	250

GTTGGTTTTAGCTTGGCCCATT	Chip strong	15833	22.509586	7.6416044	256

TTGATGCCCCGTCCTGTACACT	Chip strong	16077	20.144415	22.335653	308

GCAGGGAACTGGCTGGGCTTT	Chip strong	16084	7.1124773	22.951672	102

ACCATCTCCTGTGCCTCCAGCT	Chip strong	16520	12.522655	19.197701	320

AAGTGATACGCCTGCCTCGGCC	Chip strong	16691	9.2873106	2.0918362	2

GCCTGGCCAACATAGTGGGACC	Chip strong	16749	8.6138811	20.486101	154

TCCTGGCCATCCAGCCTGGGGA	Chip strong	16778	7.2028656	18.973217	214

TCCTCCAGAGCTTCATCCTGCC	Chip strong	16927	20.0035	5.2284846	212

GCGCCTGTGCCTCCTAA	Chip strong	17094	12.760594	23.842529	108

CTTGATTTTGTCTCTGGCCCTG	Chip strong	17456.5	9.4672995	8.272316	90

CCTGTGGTCCCTGTCTGTGCCT	Chip strong	17748	13.149311	10.342139	66

ACTTGGAACTGGCCCCTTTCAT	Chip strong	17782	14.512917	23.881441	15

TTCCCTGGGACTGGCCTGCACC	Chip strong	17948.5	9.3010607	15.061718	241

GATTACTGGTATTTGCTGGCTCC	Chip strong	13394	25.892035	5.407784	146

AGGTGGCCACAAGGTGGCTGGC	Chip strong	13621	20.378857	17.680929	334

GGCTGCTGGTCTTTCATAGTGGG	Chip strong	12604.5	21.291653	18.561375	175

CCCCTGCTGTGCTTGCATGGCT	Chip strong	12605	18.076384	11.74684	57

TGGCTTTAGTAATAAGTTTCTC	Chip strong	12660	16.773508	11.141039	226

TCTCTAGTCCTGCCTCCCC	Chip strong	12753	19.169752	7.0407801	275

TTGTCACTGCACTCCAGTCTGG	Chip strong	12372.5	9.9857264	24.029345	311

GGGAAGCTGGTCACCCACAGGC	Chip strong	12450	11.913556	20.388573	178

CTCCTTGCTGGTCTGGTGTAAT	Chip strong	12887	13.768332	6.9087734	77

TGGGTCTCTGGCCACCCCAGCC	Chip strong	12948.5	8.0436459	19.699574	228

CGGCGAGCGGGACCTGCGCCTG	Chip strong	13179	8.3394403	5.5586901	375

GCTCACAGCCTCCCCCGGCCTG	Chip strong	13198	7.8765292	3.4258959	160

TTTGGTCCCCTTCAACCAGCTA	Chip strong	13310	7.6353297	18.880299	249

TTGCTAGTGTTTGGTTGATGGT	Chip strong	13321	29.278065	21.353354	309

TGGGTCCTGGCTGAAGATCTCT	Chip strong	13345	7.4858232	22.909485	227

AGCAGAGCAGTCTCCGCTCA	Chip strong	11919	6.4712315	22.303505	327

TCTGCCTCCAGGAGCTGGCA	Chip strong	12022.5	6.4897313	19.629604	218

CTCTGATGTCTGCCCCTCACCT	Chip strong	12084	23.231821	2.7038672	83

TGGTGGAGGCGCTGCTGGCCAG	Chip strong	11424	10.211181	12.62489	233

CGCCTCCTCTCTGTCCTGATTT	Chip strong	11564	15.306285	4.1242805	372

AGGTGCTCTGTGTATGCATAGA	Chip strong	11593	19.340197	19.182079	29

GGCCGTCCCTAGAGATGGGGTT	Chip strong	11689.5	8.4446125	7.2657032	170

CATTATTCTCAGTTCTGTGCAG	Chip strong	11732.5	27.869678	16.957344	51

TGGTTTCCCTTTTGGCCTCTCC	Chip strong	10935	11.08107	6.0971227	303

CTGGCCCCTTTCATTCTGGAAG	Chip strong	11008.5	19.356289	14.29258	87

ATAGCAGCGCTGGCCCTCTGCC	Chip strong	11135.5	8.3489428	16.26886	339

TGCAGCCTCTTGTTTCAGCCCC	Chip strong	11243	17.256807	2.5227482	282

GGGTCTCTGTTGGCTTCTT	Chip strong	11264.5	7.8554482	5.5741806	128

AGCCTCTGGTCCTTTTTTCCCT	Chip strong	11308.5	17.074085	5.3993454	328

AGCTGGTTTAATATGCTGTCTG	Chip strong	11390	14.25641	8.7015753	21

CACTGCCTTGGCCACCTATCCT	Chip strong	10671	9.1234684	14.108407	346

GCCTTGGTGGTTTTGGTAGT	Chip strong	10696	15.110422	8.3110876	106

GTGGTAGCTCCAGGCTGTCTGA	Chip strong	10711	30.533655	22.150589	253

TGCTCTGATTTTTGCCCCAGCT	Chip strong	10768.5	14.230415	7.0602937	292

TCCTGGGCTTTGGCTTGTTGGG	Chip strong	10813.5	7.7058806	7.1675959	215

TCCACTGTCCCTGGCACTTTT	Chip strong	9134	6.4327211	12.8872	208

CGCCATGTCCAGCGTCTTCGGG	Chip strong	8765	20.334946	20.485155	68

CATTGCACTCCAGCCTCCCATA	Chip strong	10435	16.077471	9.6274853	53

AGAGTCTCCCTGTGTTGCCCTG	Chip strong	10467	7.4270558	12.602409	325

TCCTTCCTCTGTCAGGCAGGCC	Chip strong	10471	20.063852	2.295146	270

ACTGCACTGCAGCCTGGCCAAC	Chip strong	10584	7.3915148	12.856659	14

TTCTTCTGCCCCTTGCCTGACA	Chip strong	10593.5	16.647232	9.2061243	244

CCAGTACGTTGCTCAGCTCCTC	Chip strong	10610.5	11.484417	2.7025924	357

CGCCGCCCTCCGAGGACTCCTT	Chip strong	10614	8.6334085	6.5864415	371

TTGCTCAGGCTGGCGTGCAATG	Chip strong	9724	11.115126	19.742767	245

CCCGCGATCTCCTTGTGGCCGT	Chip strong	9728	11.945862	6.9863696	58

CACCTGGCTGGCAATTTATAAT	Chip strong	9852	8.0965796	17.484594	43

TCAGGGCTGCACTGGCTGGTCT	Chip strong	9852	10.620815	11.96568	205

TGGAGTTGGCTGCAGATGAGTC	Chip strong	9954	13.087917	15.585505	299

TGCCTAGGTCTGGCCTCCTTGG	Chip strong	10161	16.315468	2.7759731	286

GCCAGCCTCCATCCTCCCTTG	Chip strong	10191	21.391727	11.342846	149

TCCCCTCTTGGCTTGGTCCAGA	Chip strong	10285	8.0190945	16.142628	269

GGTGCCCTCTGGCTCTACTCCC	Chip strong	10302.5	7.4917507	16.076124	184

AGGGAAGGACTGCTGGGTTGGC	Chip strong	10310	6.749754	2.3204882	332

GCTGAACGAGCTGGCCAAGTTC	Chip strong	9451	6.6551905	19.321331	112

CAGCCTCTATGCCCCCGTCACC	Chip strong	9484	16.652414	11.957335	350

ACCCCGCTCCTTGCAGCCTCTG	Chip strong	9609	6.7912097	4.80404	321

CTCTTTGGTTGGTTCCTGATGC	Chip strong	9661	15.128378	18.743273	85

AATGGTCTCTTTGTTCCCTGCT	Chip strong	9183	7.6419687	3.2526188	315

AGTGTTGGCTCGGCTGGCTGCC	Chip strong	9220.5	15.521686	7.1320724	337

ATTTACATACCCAGCAGCCTCC	Chip strong	9344	14.651403	5.7202735	344

ACCTTGTGATCCACCTGCTTTG	Chip strong	9350	10.149202	4.1434402	322

TGCCAGTATCCTTCTGAGACCC	Chip strong	9374.5	18.697142	19.309006	284

ATCTCAGCTCTGCCTCCTGGGT	Chip strong	8963	12.361974	12.799247	33

TCCTCCCTCACCTCAGTCTGGG	Chip strong	8976.5	11.361602	9.0995693	213

TAGCTGAGCCGCCTGGCTGGGG	Chip strong	9026	6.8317003	8.4015751	193

CCTCTTTCACCGTGCCTGTCCC	Chip strong	8800	16.616077	5.438931	63

TCCAGGCCCTCAATCCATTTCCA	Chip strong	8934.5	13.815792	9.5553522	266

CAGGCTGGCTCCCTGAAGGTTC	Chip strong	8459.5	6.1472831	17.683357	353

TGCTCTGTTGGCTTCTTTTGTC	Chip strong	8407	17.417171	17.734081	224

CACTGTCTTCCTTTGGCTCCTC	Chip strong	8497	10.860129	11.864268	48

AGCACGGTGGGTTTGGCTGGCA	Chip strong	8532	8.91047	7.0811062	18

GTCCTCACTGGCCGCACGCTGA	Chip strong	8536	7.1346483	19.281561	188

CCAGGCTGGAGTGCAAGCAGCA	Chip strong	8552.5	11.002619	19.600433	356

CGGTGCCTCCTCCAGTGTTGCT	Chip strong	8559	10.886886	9.833169	71

GTCAGTCATTGAATGCTGGCCT	Chip strong	8592.5	23.067156	11.230301	133

CCTTTTATCCCCTAATTGGCCT	Chip strong	8596	19.616385	9.8835402	67

TGGTAGGTTGGGCAGTTC	Chip strong	8731.5	31.377066	20.530041	301

GTGTTCCTGTGCTGGATGGTCA	Chip strong	2131	11.864914	6.3784571	191

CCTCTGCACCAACCTGTCAAGA	Chip strong	2057.5	11.429537	3.11975	62

GGAGGTACTGTAGCTGGCGTT	Chip strong	1877	10.634505	9.6884193	167

GTGCTTTGCTGGAATCGAGGAA	Chip strong	1710	10.403996	8.5636625	190

AGCGTGTTGGGAGGAGCTGCAG	Chip strong	1410	9.0065594	8.8227701	19

TAGCATGGCTCTATGGAACA	Chip strong	1393	10.196934	8.9662762	260

GGCCAAGTGGATGCTGGTTTAGC	Chip strong	1351	6.3048329	7.5876508	117

AGGACCTGTAATCCCAGCACTT	Chip	1119.5	4.0140038	5.6218853	23

GTCTCGGACTCCTGATCTCAGG	Chip	1380	4.1414785	3.9894354	189

TCGCTCAGGCAGGAGTGCAGTG	Chip	1902	5.7879028	8.7315207	271

TGATCTCGTGATCTACCCGCCT	Chip	1982	5.9927278	6.810081	280

CACCTTGTGATCCACCCGCCTT	Chip	2139	5.5668392	4.7121377	44

AGTTCTCTTGCTTCAGCCTCCC	Chip	8418	11.501246	1.3339518	31

GCAGGGAACTGGCTGGGCTTTC	Chip	9142.5	5.9037857	16.801399	148

GCTCCCACTGCTGTCCTGCCAT	Chip	9433	17.716768	1.6475885	110

CCCCTCAGTTTGCTAGTATTTT	Chip	11735	24.905746	1.1986766	56

CTCGCCCCTCTCAGCCCTGCAA	Chip	14248.5	19.352268	1.4588933	79

GCCTGTCCTCTTCCGCCTGTCT	Chip	14508	12.145576	1.6282115	105

GGTTCTCAGCCTGAGCCGCCCC	Chip	18192	21.105703	1.4826102	186

CTGGCCTATCATAAGCATTTT	Chip	65516	15.111923	1.4583727	88

ACAGGCGATCCACCCGCCTCAG	Chip	2228	5.9650521	8.9491081	318

GAACTTGTGATCCGCCCACCTT	Chip	2483	4.4610376	7.0900927	93

GACCTTGTGATCCACCTGTTTT	Chip	2612	4.8775668	12.335071	98

CTCTGAGTCCTGCACTCACCCG	Chip	2770	6.7869315	1.284364	82

CTGCAGCCTCCACTTTCTGGGC	Chip	2839	4.7054248	13.918253	380

GTGTTGTCGCTGGGTTTTGAGGG	Chip	3030	4.5279474	3.9595523	254

TAGGAGGATTGCTTGTGGCCAG	Chip	3154.5	4.6519237	4.9273152	194

CGGTGGGTGCTTCAGGCGGTGG	Chip	3999	5.0099111	5.715847	376

GTGACTGTGGGTTTCTGGTTCC	Chip	4025.5	5.8571658	7.4026732	136

GCTGCTGGGCCATTTGTTGG	Chip	4101	7.7621112	1.3319389	114

GCAGGCTCTGGCTTATTCTGGG	Chip	4399	4.4706116	13.904231	101

GCGGGCGGCTTCATCTTGCCCT	Chip	5038	5.1213508	7.6892729	158

TCCCAGCTCCTGGGCCCCACAG	Chip	5372.5	4.9255114	7.1915674	267

ATCTTTTATCACTCCCACTGCT	Chip	5396	5.4679914	11.567021	340

GATGGGTTTGTTGGAGAGGTC	Chip	5425.5	4.8749881	17.533426	144

GTGACCTGGCCGCCTAAACCCA	Chip	5941.5	5.6531525	18.527802	135

AAGACACCAGAGACTGGCCTCA	Chip	6306	5.8909965	5.1631103	314

TCCTCAGCTTGGCCACGGAGTT	Chip	6478.5	5.8972673	17.989834	211

TGTCTCCCCACTGGTCTTCCAG	Chip	7039	5.6089306	15.167439	235

AAACTGCTTCCTTGGCCT	Chip	7436	5.6282043	5.6413546	312



ROW#	INFECTION NAME	SEQ ID NOs OF GAMS ASSOCIATED WITH INFECTION

2	Bordetella pertussis	1, 6, 10, 11, 12, 13, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33,
		34, 37, 41, 42, 43, 44, 47, 48, 49, 50, 52, 53, 54, 55, 57, 58, 59, 60, 63, 65,
		66, 67, 68, 69, 70, 71, 75, 76, 77, 79, 84, 86, 87, 88, 89, 91, 94, 96, 97, 99,
		100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
		117, 119, 120, 121, 122, 123, 125, 126, 127, 130, 131, 132, 133, 137, 138, 139,
		140, 141, 142, 145, 147, 149, 150, 151, 154, 155, 156, 157, 158, 160, 161, 162,
		164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181,
		183, 184, 185, 188, 191, 195, 196, 197, 204, 205, 211, 212, 214, 215, 216, 219,
		220, 222, 225, 228, 230, 231, 233, 237, 239, 241, 242, 243, 244, 250, 251, 253,
		262, 264, 265, 266, 268, 271, 272, 274, 276, 277, 280, 281, 282, 284, 285, 287,
		288, 289, 290, 293, 294, 296, 297, 299, 300, 301, 302, 304, 306, 308, 310, 312,
		317, 318, 321, 322, 324, 326, 327, 329, 330, 332, 333, 334, 335, 336, 339, 340,
		342, 343, 345, 348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 360, 361, 362,
		364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 378, 380, 381,
		382, 383, 384, 385 and 49788-55666.
3	Brucella suis 1330	1, 6, 10, 11, 12, 13, 14, 16, 18, 19, 21, 23, 27, 32, 35, 37, 39, 40, 42, 47,
		48, 49, 50, 52, 53, 58, 62, 63, 65, 68, 70, 71, 77, 79, 80, 85, 86, 89, 90, 98,
		102, 105, 107, 108, 109, 111, 112, 114, 115, 119, 120, 121, 122, 123, 124, 125,
		126, 132, 138, 141, 142, 143, 150, 151, 152, 154, 155, 156, 157, 158, 160, 161,
		162, 164, 166, 168, 171, 172, 173, 175, 176, 177, 180, 181, 183, 185, 186, 190,
		195, 198, 199, 200, 201, 205, 207, 211, 212, 214, 215, 217, 218, 219, 220, 221,
		222, 225, 229, 230, 231, 233, 236, 237, 240, 241, 243, 244, 250, 251, 256, 258,
		263, 264, 265, 266, 270, 277, 279, 280, 281, 282, 285, 287, 289, 290, 293, 294,
		295, 297, 300, 302, 303, 306, 308, 310, 312, 315, 318, 319, 320, 321, 330, 331,
		333, 334, 335, 342, 343, 347, 348, 349, 353, 354, 356, 357, 360, 361, 364, 365,
		366, 368, 369, 370, 371, 373, 374, 375, 377, 381, 382, 384 and 55667-60259.
4	Chlamydia trachomatis	2, 3, 4, 6, 7, 8, 9, 10, 13, 14, 16, 18, 19, 20, 21, 22, 25, 26, 27, 30, 31, 32,
		33, 36, 37, 38, 40, 45, 46, 47, 48, 49, 51, 52, 55, 62, 63, 64, 67, 73, 74, 75,
		78, 81, 82, 84, 85, 86, 87, 88, 91, 94, 95, 98, 99, 104, 105, 106, 111, 113,
		116, 122, 124, 126, 128, 132, 133, 136, 138, 146, 148, 149, 152, 154, 155, 156,
		157, 160, 164, 166, 167, 177, 179, 180, 181, 187, 188, 190, 192, 194, 198, 199,
		200, 205, 207, 208, 209, 210, 211, 213, 214, 217, 218, 222, 224, 225, 226, 229,
		232, 233, 235, 236, 239, 241, 242, 243, 244, 245, 248, 251, 252, 253, 254, 256,
		257, 259, 262, 264, 265, 269, 270, 271, 272, 273, 274, 278, 279, 287, 288, 289,
		293, 295, 296, 297, 298, 299, 302, 303, 305, 306, 309, 311, 312, 316, 318, 319,
		320, 322, 323, 324, 325, 326, 327, 328, 330, 332, 333, 335, 338, 340, 341, 343,
		344, 345, 348, 349, 350, 353, 354, 356, 363, 373, 384 and 60260-67437.
5	Chlamydophila pneumoniae	25, 27, 33, 46, 55, 62, 73, 105, 152, 160, 166, 177, 179, 180, 190, 205, 208,
	AR39	213, 214, 218, 236, 242, 244, 262, 271, 274, 298, 323, 325, 327, 345, 353, 356
		and 67438-68147.
6	Chlamydophila pneumoniae	3, 5, 6, 8, 9, 10, 13, 17, 20, 21, 22, 23, 25, 27, 28, 31, 32, 33, 37, 39, 45,
	CWL029	46, 47, 48, 50, 52, 55, 62, 63, 64, 66, 67, 69, 73, 74, 82, 84, 85, 88, 89, 90,
		91, 92, 95, 101, 102, 104, 105, 111, 114, 124, 125, 126, 128, 143, 146, 148,
		152, 159, 160, 161, 164, 165, 166, 168, 175, 176, 177, 178, 179, 180, 181, 187,
		189, 190, 192, 194, 201, 203, 205, 207, 208, 209, 212, 213, 214, 217, 218, 221,
		223, 224, 227, 232, 233, 234, 236, 238, 239, 241, 242, 243, 244, 245, 247, 248,
		252, 257, 258, 259, 260, 262, 263, 271, 272, 274, 275, 279, 281, 282, 283, 286,
		289, 295, 297, 298, 299, 302, 305, 306, 309, 311, 312, 314, 319, 323, 324, 325,
		326, 327, 330, 333, 338, 340, 343, 344, 345, 346, 348, 349, 350, 352, 353, 354,
		356, 363, 377, 382, 383, 384 and 68148-75439.
7	Chlamydophila pneumoniae	3, 5, 6, 8, 9, 10, 17, 20, 21, 22, 23, 25, 27, 31, 32, 33, 37, 39, 45, 46, 47,
	J138	50, 52, 55, 62, 63, 64, 66, 67, 69, 73, 74, 82, 84, 85, 88, 89, 90, 92, 95, 101,
		102, 104, 105, 111, 114, 125, 126, 128, 143, 146, 148, 152, 159, 160, 161, 164,
		165, 166, 168, 175, 176, 177, 178, 179, 180, 181, 187, 189, 190, 192, 194, 201,
		203, 205, 207, 208, 209, 212, 213, 214, 217, 218, 221, 223, 224, 227, 232, 233,
		234, 236, 238, 239, 241, 242, 243, 244, 245, 247, 248, 252, 257, 259, 260, 262,
		263, 271, 272, 274, 275, 279, 281, 282, 283, 286, 289, 295, 297, 298, 299, 302,
		305, 306, 309, 311, 312, 314, 319, 323, 325, 326, 327, 330, 333, 338, 340, 343,
		344, 345, 346, 348, 349, 350, 352, 353, 354, 356, 363, 377, 382, 383, 384 and
		75440-82241.
8	Chlamydophila pneumoniae	20, 21, 22, 25, 27, 31, 33, 45, 46, 50, 55, 62, 64, 73, 82, 89, 92, 104, 105,
	TW-183	126, 143, 146, 152, 160, 161, 166, 175, 177, 178, 179, 180, 187, 190, 201, 205,
		208, 209, 212, 213, 214, 217, 218, 221, 232, 236, 239, 242, 244, 248, 257, 262,
		263, 271, 272, 274, 275, 279, 282, 289, 298, 299, 302, 306, 312, 323, 325, 327,
		338, 340, 345, 346, 350, 352, 353, 356, 363, 382 and 82242-85213.
9	Coxiella burnetii RSA	1, 3, 5, 6, 7, 8, 10, 13, 22, 25, 27, 33, 36, 38, 40, 42, 45, 46, 48, 51, 52,
	493	55, 62, 67, 73, 78, 80, 81, 84, 91, 105, 111, 116, 124, 126, 132, 141, 142, 146,
		147, 152, 158, 160, 164, 166, 177, 179, 180, 186, 187, 190, 205, 208, 213, 214,
		218, 227, 229, 232, 234, 236, 239, 241, 242, 244, 247, 248, 249, 252, 256, 259,
		262, 268, 271, 272, 274, 279, 280, 281, 282, 285, 298, 299, 300, 303, 305, 306,
		307, 312, 315, 316, 320, 323, 324, 325, 326, 327, 333, 340, 344, 345, 353, 354,
		356, 365, 373, 374, 376, 379, 385 and 85214-90622.
10	Escherichia coli CFT	1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 25, 26,
	073	27, 28, 30, 31, 33, 34, 35, 36, 37, 39, 40, 42, 43, 45, 46, 47, 48, 49, 50, 51,
		52, 53, 55, 56, 57, 58, 59, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75,
		76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
		97, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,
		115, 116, 119, 120, 121, 122, 123, 124, 125, 126, 129, 131, 132, 133, 135, 136,
		137, 138, 140, 141, 142, 143, 145, 146, 147, 148, 152, 154, 155, 156, 157, 158,
		160, 161, 162, 163, 164, 165, 166, 167, 168, 171, 173, 174, 175, 176, 177, 179,
		180, 181, 182, 184, 185, 186, 190, 191, 192, 193, 195, 196, 197, 203, 204, 205,
		206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 221, 222,
		223, 224, 225, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242,
		243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,
		260, 261, 262, 265, 266, 267, 268, 270, 271, 272, 274, 276, 277, 278, 279, 280,
		281, 282, 283, 284, 285, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,
		299, 300, 301, 302, 303, 305, 306, 307, 308, 309, 310, 311, 312, 314, 315, 316,
		317, 318, 321, 322, 323, 324, 325, 326, 327, 329, 331, 332, 333, 334, 335, 336,
		337, 338, 339, 340, 343, 344, 345, 347, 348, 349, 350, 351, 352, 353, 354, 355,
		356, 357, 358, 359, 360, 361, 364, 365, 367, 368, 369, 370, 372, 373, 374, 375,
		376, 378, 380, 381, 382, 383, 384, 385 and 90623-103607.
11	Haemophilus influenzae	2, 3, 5, 6, 7, 8, 9, 10, 13, 15, 19, 20, 21, 22, 25, 26, 27, 30, 31, 32, 33, 34,
	Rd	37, 38, 40, 41, 45, 46, 48, 49, 50, 51, 52, 53, 55, 62, 63, 64, 66, 67, 68, 73,
		78, 81, 83, 84, 85, 88, 90, 91, 92, 98, 101, 105, 106, 111, 116, 117, 119, 122,
		123, 124, 125, 126, 134, 138, 144, 146, 149, 151, 152, 155, 156, 160, 161, 164,
		165, 166, 169, 171, 172, 174, 176, 177, 179, 180, 183, 190, 197, 198, 199, 200,
		201, 203, 205, 207, 208, 211, 213, 214, 218, 221, 223, 226, 228, 229, 234, 236,
		239, 240, 242, 244, 247, 248, 251, 254, 255, 256, 259, 262, 263, 264, 271, 272,
		274, 277, 279, 281, 282, 283, 295, 296, 299, 302, 305, 306, 308, 311, 312, 313,
		316, 317, 318, 319, 322, 323, 324, 325, 326, 327, 329, 333, 335, 338, 339, 340,
		343, 344, 345, 348, 351, 353, 354, 356, 365, 368, 371, 375, 377, 379, 380, 385
		and 103608-111433.
12	Leptospira interrogans	1, 3, 5, 7, 8, 10, 13, 19, 22, 25, 32, 38, 39, 41, 48, 49, 52, 67, 71, 73, 84,
	serovar lai str.	85, 90, 91, 93, 95, 117, 124, 128, 164, 174, 178, 179, 187, 190, 192, 193, 203,
	56601	207, 225, 226, 227, 229, 238, 244, 247, 250, 256, 257, 258, 259, 262, 272, 279,
		295, 298, 299, 303, 306, 307, 316, 324, 327, 333, 338, 340, 344, 348, 376, 379,
		384 and 111434-116384.
13	Listeria monocytogenes	5, 6, 7, 8, 9, 10, 13, 22, 36, 40, 48, 52, 67, 84, 90, 91, 95, 114, 116, 147,
	EGD-e	185, 214, 244, 247, 248, 253, 254, 259, 262, 272, 276, 279, 299, 306, 308, 324,
		333, 340, 355, 382 and 116385-119434.
14	Mycobacterium avium	1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
	subsp. paratuberculosis	25, 26, 27, 29, 31, 32, 33, 34, 37, 42, 43, 44, 45, 46, 47, 50, 51, 53, 54, 55,
		58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79,
		84, 86, 87, 88, 89, 90, 91, 94, 96, 97, 99, 100, 101, 102, 103, 105, 107, 108,
		109, 110, 111, 112, 113, 114, 115, 116, 119, 120, 121, 122, 123, 125, 127, 130,
		131, 132, 133, 135, 137, 138, 139, 140, 142, 143, 144, 146, 147, 148, 149, 150,
		151, 152, 154, 155, 156, 157, 158, 160, 161, 162, 164, 165, 166, 167, 168, 170,
		171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 183, 184, 185, 188, 189, 190,
		191, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 207, 210, 211, 214, 215,
		216, 218, 219, 220, 222, 225, 226, 230, 231, 233, 234, 236, 237, 239, 241, 242,
		243, 244, 245, 248, 250, 251, 252, 253, 254, 257, 262, 263, 264, 265, 266, 268,
		271, 272, 274, 277, 278, 280, 281, 282, 283, 285, 287, 288, 289, 290, 291, 292,
		293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 305, 306, 310, 312, 313,
		314, 318, 320, 321, 323, 324, 325, 327, 329, 331, 332, 333, 334, 335, 336, 337,
		341, 342, 345, 346, 347, 349, 351, 352, 353, 355, 356, 357, 358, 360, 361, 362,
		364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380,
		381, 382, 383, 384 and 119435-127918.
15	Mycobacterium bovis	1, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
	subsp bovis AF2122/97	26, 27, 28, 29, 31, 32, 33, 36, 37, 39, 41, 42, 43, 45, 46, 47, 48, 50, 51, 52,
		53, 54, 55, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 73, 75, 76,
		77, 78, 79, 80, 83, 84, 86, 87, 88, 89, 90, 91, 93, 96, 97, 99, 100, 101, 102,
		103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
		119, 120, 121, 122, 123, 125, 127, 130, 131, 132, 133, 134, 135, 137, 138, 139,
		141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157,
		158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175,
		176, 177, 178, 179, 180, 181, 183, 184, 185, 188, 189, 190, 191, 193, 194, 195,
		196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212,
		213, 214, 215, 216, 218, 219, 220, 222, 225, 230, 231, 233, 236, 237, 239, 240,
		241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257, 261, 262,
		263, 264, 265, 266, 267, 268, 270, 271, 273, 276, 277, 278, 280, 281, 282, 283,
		285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 296, 297, 299, 300, 302, 303,
		304, 305, 306, 308, 310, 312, 313, 314, 315, 318, 320, 321, 322, 323, 324, 325,
		326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 341, 342, 345, 346, 347,
		348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 360, 361, 362, 364, 365, 366,
		367, 369, 370, 371, 372, 373, 374, 375, 376, 378, 380, 381, 382, 383, 384, 385
		and 127919-137561.
16	Mycobacterium leprae	3, 4, 5, 6, 7, 12, 13, 14, 15, 18, 19, 21, 22, 23, 24, 26, 29, 31, 32, 33, 36,
		37, 39, 41, 42, 43, 45, 46, 47, 48, 49, 50, 53, 54, 57, 59, 62, 65, 68, 69, 70,
		71, 73, 74, 75, 76, 78, 81, 83, 84, 86, 90, 94, 96, 98, 101, 103, 106, 107, 109,
		110, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 123, 131, 133, 134, 135,
		137, 142, 143, 144, 145, 146, 147, 149, 154, 156, 157, 158, 159, 161, 162, 163,
		165, 166, 167, 171, 172, 173, 174, 175, 176, 179, 183, 184, 185, 187, 188, 189,
		190, 193, 196, 197, 198, 199, 200, 201, 202, 204, 205, 206, 211, 212, 214, 215,
		216, 218, 219, 220, 221, 223, 224, 225, 228, 230, 231, 232, 233, 234, 235, 236,
		237, 241, 242, 243, 245, 249, 250, 251, 253, 254, 256, 258, 261, 263, 265, 267,
		268, 269, 271, 274, 276, 277, 280, 281, 284, 288, 289, 290, 291, 293, 294, 295,
		296, 297, 299, 300, 301, 302, 303, 305, 306, 307, 309, 310, 311, 312, 313, 314,
		315, 318, 320, 321, 323, 324, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337,
		338, 339, 340, 341, 343, 345, 346, 347, 348, 349, 353, 355, 356, 357, 358, 360,
		361, 364, 365, 368, 369, 370, 371, 372, 374, 375, 376, 377, 378, 380, 381, 382,
		383 and 137562-144598.
17	Mycobacterium tuberculosis	4, 5, 6, 7, 10, 13, 17, 20, 22, 23, 24, 25, 27, 31, 32, 33, 45, 46, 51, 53, 55,
	CDC1551	62, 67, 69, 73, 84, 88, 90, 91, 99, 100, 102, 103, 105, 107, 113, 114, 116, 120,
		137, 143, 146, 148, 149, 152, 155, 156, 160, 161, 165, 166, 168, 177, 179, 180,
		185, 190, 198, 199, 200, 203, 205, 207, 208, 211, 213, 214, 215, 216, 218, 219,
		225, 233, 236, 239, 242, 244, 257, 262, 264, 271, 272, 274, 281, 282, 289, 291,
		292, 294, 299, 303, 305, 306, 312, 313, 323, 324, 325, 327, 329, 332, 333, 337,
		341, 345, 346, 352, 353, 356, 381, 383 and 144599-146806.
18	Mycobacterium tuberculosis	1, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
	H37Rv	25, 26, 27, 28, 29, 31, 32, 33, 37, 39, 41, 42, 43, 45, 46, 47, 48, 50, 51, 52,
		53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 75,
		76, 77, 78, 79, 80, 83, 84, 86, 87, 88, 89, 90, 91, 93, 94, 96, 97, 99, 100,
		101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
		118, 119, 120, 121, 122, 123, 125, 127, 130, 131, 132, 133, 134, 135, 137, 138,
		139, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
		156, 157, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 171, 172, 173,
		174, 175, 176, 177, 178, 179, 180, 181, 183, 184, 185, 188, 189, 190, 191, 194,
		195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 210, 211,
		212, 213, 214, 215, 216, 218, 219, 220, 222, 225, 230, 231, 233, 234, 236, 237,
		239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257,
		261, 262, 263, 264, 265, 266, 267, 268, 270, 271, 272, 273, 274, 276, 277, 278,
		280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 296,
		297, 298, 299, 300, 302, 303, 304, 305, 306, 308, 310, 312, 313, 314, 315, 318,
		320, 321, 323, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337,
		341, 342, 345, 346, 347, 348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 360,
		361, 362, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 378,
		380, 381, 382, 383, 384, 385 and 146807-155497.
19	Neisseria meningitidis	56, 142, 218, 287, 316, 327, 351, 355, 365, 381 and 155498-155833.
	MC58
20	Neisseria meningitidis	1, 6, 7, 8, 10, 12, 15, 17, 21, 22, 26, 28, 30, 37, 39, 40, 45, 49, 52, 56, 58,
	Z2491	60, 62, 63, 67, 70, 76, 86, 89, 90, 91, 96, 98, 102, 103, 105, 107, 108, 109,
		111, 112, 113, 114, 115, 122, 123, 124, 125, 126, 127, 133, 138, 141, 142, 143,
		145, 147, 148, 149, 152, 157, 158, 164, 165, 166, 170, 171, 175, 176, 178, 181,
		183, 187, 189, 197, 203, 217, 218, 219, 220, 221, 222, 225, 229, 230, 231, 237,
		239, 243, 245, 247, 248, 251, 253, 254, 256, 257, 258, 259, 264, 265, 268, 273,
		281, 282, 283, 285, 287, 289, 290, 293, 294, 295, 297, 300, 302, 306, 308, 314,
		315, 316, 319, 321, 322, 325, 327, 329, 332, 333, 334, 338, 340, 341, 344, 346,
		348, 349, 350, 351, 354, 355, 356, 365, 371, 372, 375, 376, 380, 381, 382, 384
		and 155834-160603.
21	Pseudomonas aeruginosa	1, 2, 6, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
	PA01	29, 30, 31, 33, 34, 35, 36, 37, 41, 42, 43, 45, 46, 48, 49, 50, 52, 54, 55, 56,
		57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 76, 77, 78, 79, 81,
		82, 83, 84, 86, 87, 88, 89, 90, 91, 94, 95, 96, 97, 99, 100, 101, 102, 103, 105,
		106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122,
		123, 124, 125, 126, 127, 130, 131, 134, 137, 138, 139, 140, 141, 142, 144, 147,
		149, 150, 151, 152, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164, 165, 166,
		167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 183, 184,
		185, 188, 190, 192, 193, 194, 195, 196, 197, 202, 204, 205, 208, 210, 211, 212,
		213, 214, 215, 216, 218, 220, 222, 225, 228, 229, 230, 231, 232, 233, 236, 237,
		241, 242, 243, 244, 250, 251, 253, 258, 262, 264, 265, 266, 267, 268, 270, 271,
		272, 273, 274, 276, 277, 280, 281, 282, 283, 285, 286, 287, 288, 289, 290, 291,
		292, 293, 294, 296, 297, 298, 299, 300, 301, 302, 306, 312, 314, 318, 319, 320,
		321, 323, 324, 325, 327, 329, 330, 331, 333, 334, 335, 336, 339, 340, 341, 342,
		343, 345, 347, 348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 360, 361, 362,
		364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 378, 380, 381,
		382, 383, 384 and 160604-170274.
22	Pseudomonas putida KT2440	1, 5, 7, 9, 10, 11, 12, 13, 14, 16, 18, 19, 23, 24, 25, 26, 27, 28, 29, 31, 33,
		34, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 57,
		58, 59, 61, 64, 65, 66, 68, 69, 70, 71, 73, 76, 84, 85, 86, 88, 89, 91, 94, 98,
		99, 101, 102, 103, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 117,
		118, 119, 120, 121, 122, 123, 125, 126, 131, 132, 133, 134, 135, 137, 138, 140,
		141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
		157, 158, 159, 160, 161, 162, 163, 164, 166, 167, 168, 171, 172, 173, 174, 175,
		176, 177, 179, 180, 181, 183, 184, 185, 187, 190, 191, 193, 195, 196, 197, 202,
		204, 205, 207, 211, 212, 214, 215, 216, 220, 221, 222, 225, 228, 229, 230, 231,
		232, 233, 234, 236, 237, 240, 241, 242, 243, 244, 248, 250, 251, 253, 255, 258,
		264, 265, 266, 267, 270, 271, 272, 274, 276, 277, 280, 281, 282, 283, 284, 285,
		287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,
		303, 304, 305, 306, 308, 310, 312, 313, 314, 316, 317, 318, 320, 321, 322, 323,
		324, 327, 329, 333, 334, 335, 336, 337, 342, 343, 345, 346, 347, 348, 349, 350,
		351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 364, 365, 366, 367, 368, 369,
		370, 371, 373, 374, 375, 376, 377, 378, 380, 381, 382, 383, 384, 385 and
		170275-178543.
23	Rickettsia prowazekii	2, 10, 13, 25, 27, 31, 33, 45, 46, 48, 52, 55, 62, 67, 71, 73, 75, 81, 84, 91,
		95, 99, 100, 105, 113, 124, 131, 152, 154, 155, 156, 157, 160, 162, 166, 177,
		179, 180, 181, 190, 192, 204, 205, 208, 213, 214, 217, 218, 222, 231, 236, 239,
		242, 244, 262, 265, 270, 271, 272, 274, 278, 287, 288, 289, 293, 294, 299, 305,
		306, 323, 324, 325, 327, 333, 334, 340, 345, 353, 356, 373, 381 and
		178544-179914.
24	Salmonella enterica	1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26,
	enterica serovar Typhi	27, 28, 30, 31, 32, 33, 35, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, 50, 51,
		52, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75,
		77, 79, 80, 81, 83, 84, 86, 88, 89, 90, 91, 92, 94, 95, 98, 99, 100, 101, 102,
		105, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 119, 120, 121, 122, 123,
		124, 125, 126, 127, 129, 131, 132, 133, 135, 136, 137, 138, 142, 143, 144, 145,
		146, 147, 148, 150, 152, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163, 164,
		165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 182, 183, 185,
		187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,
		203, 204, 205, 206, 208, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,
		223, 225, 226, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,
		242, 243, 244, 245, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 260, 261,
		262, 263, 265, 266, 269, 270, 271, 272, 274, 276, 277, 278, 280, 281, 282, 283,
		284, 285, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,
		301, 302, 303, 304, 305, 306, 308, 311, 312, 314, 315, 318, 319, 323, 324, 325,
		327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 340, 341, 342, 343,
		344, 345, 346, 347, 348, 349, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361,
		364, 365, 366, 367, 369, 370, 371, 373, 374, 375, 376, 378, 379, 380, 381, 382,
		383, 384, 385 and 179915-190940.
25	Salmonella enterica	1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 25,
	enterica serovar Typhi	26, 27, 28, 30, 31, 32, 33, 35, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, 50,
	Ty2	51, 52, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
		75, 77, 79, 80, 81, 83, 84, 85, 86, 88, 89, 90, 91, 94, 95, 98, 99, 100, 101,
		102, 105, 106, 107, 108, 109, 111, 112, 113, 114, 115, 116, 119, 120, 121, 122,
		123, 124, 125, 126, 127, 129, 131, 132, 133, 135, 136, 137, 138, 142, 143, 144,
		145, 146, 147, 148, 150, 152, 153, 154, 155, 156, 157, 158, 160, 161, 162, 163,
		164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 182, 183,
		185, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,
		202, 203, 204, 205, 206, 208, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,
		222, 223, 225, 226, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,
		241, 242, 243, 244, 245, 247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 260,
		261, 262, 263, 265, 266, 269, 270, 271, 272, 274, 276, 277, 278, 280, 281, 282,
		283, 284, 285, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
		300, 301, 302, 303, 304, 305, 306, 308, 311, 312, 314, 315, 318, 319, 323, 324,
		325, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,
		342, 343, 344, 345, 346, 347, 348, 349, 351, 352, 353, 354, 355, 356, 357, 358,
		360, 361, 364, 365, 366, 367, 369, 370, 371, 373, 374, 375, 376, 378, 379, 380,
		381, 382, 383, 384, 385 and 190941-201927.
26	Salmonella typhimurium	1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24,
	LT2	25, 26, 27, 28, 29, 30, 31, 32, 33, 36, 37, 38, 39, 42, 43, 45, 46, 47, 48, 49,
		50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
		72, 73, 75, 77, 79, 82, 83, 84, 86, 88, 89, 90, 91, 94, 95, 96, 100, 101, 102,
		103, 104, 105, 107, 108, 109, 111, 112, 113, 114, 115, 116, 119, 120, 121, 122,
		123, 124, 125, 126, 127, 129, 131, 132, 133, 135, 137, 138, 142, 143, 144, 145,
		146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 160, 161, 162, 163,
		164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181,
		182, 183, 185, 187, 188, 189, 190, 192, 193, 194, 195, 196, 197, 198, 199, 200,
		201, 202, 203, 204, 205, 206, 207, 208, 211, 212, 213, 214, 215, 216, 217, 218,
		219, 220, 221, 222, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
		237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 249, 250, 251, 252, 253,
		255, 256, 257, 258, 260, 261, 262, 263, 266, 267, 268, 270, 271, 272, 273, 274,
		275, 276, 279, 280, 281, 282, 283, 285, 287, 288, 289, 290, 291, 292, 293, 294,
		296, 297, 298, 299, 300, 302, 303, 306, 307, 308, 309, 310, 311, 312, 314, 315,
		317, 318, 319, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,
		336, 337, 338, 340, 341, 342, 343, 344, 345, 347, 348, 349, 350, 351, 352, 353,
		354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 368, 369, 370,
		371, 373, 374, 375, 376, 379, 380, 381, 382, 383, 384, 385 and 201928-215605.
27	Shigella flexneri 2a	1, 2, 5, 6, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27,
	str. 2457T	28, 29, 30, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 46, 47, 48, 49, 50, 51,
		52, 54, 55, 56, 57, 58, 59, 62, 63, 65, 66, 67, 68, 69, 70, 71, 73, 76, 78, 80,
		83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 97, 99, 101, 102, 103, 104, 105,
		107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 119, 120, 121, 122, 123,
		124, 125, 126, 129, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 142, 143,
		145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 160, 161, 162,
		163, 164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 182,
		184, 185, 187, 190, 191, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,
		207, 208, 212, 213, 214, 216, 218, 220, 221, 222, 223, 224, 225, 229, 230, 231,
		232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 247, 248, 250,
		251, 252, 253, 254, 255, 256, 257, 260, 261, 262, 263, 265, 268, 270, 271, 272,
		274, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 287, 288, 289, 290, 291,
		292, 293, 295, 296, 297, 298, 299, 300, 301, 302, 304, 306, 307, 308, 309, 310,
		311, 312, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 327, 328, 329,
		331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 344, 345, 346, 347,
		348, 349, 350, 351, 352, 353, 354, 356, 357, 358, 359, 360, 361, 362, 364, 365,
		366, 367, 368, 369, 371, 373, 374, 375, 376, 379, 380, 381, 382, 383, 384, 385
		and 215606-226197.
28	Shigella flexneri 2a	1, 2, 5, 6, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27,
	str. 301	28, 29, 30, 32, 33, 35, 36, 37, 39, 40, 41, 42, 43, 46, 47, 48, 49, 50, 51, 52,
		54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 76, 77, 78,
		80, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 99, 101, 102, 103,
		104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 119, 120, 121, 122,
		123, 124, 125, 126, 129, 132, 133, 134, 135, 136, 137, 138, 141, 142, 143, 144,
		145, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, 160, 161,
		162, 163, 164, 165, 166, 167, 168, 171, 172, 173, 174, 175, 176, 177, 179, 180,
		181, 182, 184, 185, 187, 190, 191, 195, 196, 197, 198, 199, 200, 201, 202, 203,
		205, 207, 208, 210, 212, 213, 214, 216, 217, 218, 220, 221, 222, 223, 224, 225,
		229, 230, 231, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
		247, 248, 250, 251, 252, 253, 254, 255, 256, 257, 260, 262, 263, 264, 265, 266,
		268, 269, 270, 271, 272, 274, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285,
		287, 288, 289, 290, 291, 292, 293, 295, 296, 297, 298, 299, 300, 301, 302, 304,
		306, 308, 309, 311, 312, 314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 327,
		328, 329, 331, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 344, 345, 346,
		347, 348, 349, 350, 351, 352, 353, 354, 356, 357, 358, 359, 360, 361, 362, 364,
		365, 366, 367, 368, 369, 371, 373, 374, 375, 376, 378, 379, 380, 381, 382, 383,
		384, 385 and 226198-237003.
29	Staphylococcus aureus	2, 5, 7, 8, 9, 10, 13, 16, 19, 22, 25, 27, 31, 32, 33, 35, 36, 38, 39, 40, 41,
	subsp. aureus Mu50	45, 46, 47, 48, 50, 51, 52, 55, 62, 63, 67, 71, 73, 81, 83, 84, 85, 90, 91, 92,
		93, 95, 98, 100, 101, 105, 106, 111, 113, 116, 119, 120, 124, 131, 133, 138,
		139, 146, 147, 149, 152, 153, 156, 160, 161, 162, 165, 166, 169, 171, 172, 174,
		177, 179, 180, 181, 190, 192, 203, 204, 205, 207, 208, 213, 214, 217, 218, 222,
		228, 231, 232, 236, 238, 240, 242, 244, 245, 247, 248, 252, 254, 256, 259, 261,
		262, 270, 271, 272, 274, 275, 287, 293, 294, 299, 301, 302, 305, 306, 308, 309,
		311, 316, 317, 323, 324, 325, 326, 327, 332, 333, 334, 335, 337, 339, 340, 342,
		343, 344, 345, 346, 348, 349, 351, 353, 354, 356, 363, 365, 368, 371, 375, 379,
		381 and 237004-244310.
30	Staphylococcus aureus	2, 5, 7, 8, 10, 13, 16, 19, 22, 25, 27, 30, 31, 32, 33, 38, 39, 40, 41, 45, 46,
	subsp. aureus MW2	47, 48, 50, 51, 52, 55, 62, 63, 67, 71, 72, 73, 78, 81, 83, 84, 90, 91, 92, 93,
		95, 98, 100, 101, 105, 106, 109, 111, 113, 117, 119, 120, 124, 126, 128, 130,
		131, 133, 134, 138, 139, 143, 149, 152, 153, 156, 160, 161, 162, 166, 169, 171,
		172, 174, 177, 179, 180, 181, 182, 190, 192, 203, 204, 205, 207, 208, 213, 214,
		217, 218, 222, 228, 231, 232, 236, 238, 242, 244, 247, 248, 252, 254, 256, 257,
		259, 261, 262, 271, 272, 274, 279, 287, 293, 294, 295, 299, 301, 302, 306, 307,
		308, 309, 315, 316, 323, 324, 325, 326, 327, 332, 333, 334, 335, 337, 338, 339,
		342, 343, 344, 345, 346, 348, 350, 351, 353, 356, 363, 365, 368, 371, 375, 379,
		381 and 244311-250683.
31	Staphylococcus aureus	2, 5, 7, 8, 9, 10, 13, 16, 19, 22, 25, 27, 31, 32, 33, 35, 36, 38, 39, 40, 41,
	subsp. aureus N315	45, 46, 47, 48, 50, 51, 52, 55, 62, 63, 67, 71, 73, 81, 83, 84, 85, 90, 91, 92,
		93, 95, 98, 100, 101, 105, 106, 111, 113, 117, 119, 120, 124, 131, 133, 134,
		138, 139, 143, 146, 147, 149, 152, 153, 156, 160, 161, 162, 166, 169, 171, 172,
		174, 177, 179, 180, 181, 190, 192, 203, 204, 205, 207, 208, 213, 214, 217, 218,
		222, 226, 228, 231, 232, 236, 238, 240, 242, 244, 245, 247, 248, 252, 254, 256,
		259, 260, 261, 262, 270, 271, 272, 274, 275, 279, 287, 293, 294, 299, 301, 302,
		305, 306, 307, 308, 309, 311, 316, 317, 323, 324, 325, 326, 327, 332, 333, 334,
		335, 337, 339, 340, 342, 343, 344, 345, 346, 348, 349, 351, 353, 354, 356, 363,
		365, 368, 371, 375, 379, 381 and 250684-257140.
32	Streptococcus pneumoniae	2, 3, 5, 6, 10, 13, 14, 17, 20, 21, 22, 23, 25, 26, 27, 30, 31, 32, 33, 35, 36,
	R6	37, 38, 39, 40, 41, 46, 47, 48, 49, 50, 52, 55, 56, 62, 63, 67, 73, 77, 81, 83,
		84, 85, 87, 90, 91, 92, 94, 95, 100, 101, 102, 105, 106, 111, 112, 114, 115,
		116, 117, 119, 123, 124, 126, 133, 136, 138, 143, 145, 146, 147, 149, 152, 156,
		160, 161, 164, 166, 168, 169, 171, 172, 174, 175, 176, 177, 179, 180, 190, 192,
		203, 204, 205, 208, 209, 213, 214, 217, 218, 223, 226, 228, 229, 232, 233, 235,
		236, 238, 239, 242, 244, 245, 246, 247, 248, 249, 252, 255, 256, 257, 258, 259,
		260, 261, 262, 264, 268, 271, 272, 274, 279, 282, 283, 284, 287, 295, 296, 297,
		298, 299, 300, 302, 303, 305, 306, 307, 309, 311, 312, 314, 315, 316, 320, 321,
		323, 324, 325, 326, 327, 329, 333, 335, 338, 340, 341, 344, 345, 348, 350, 351,
		352, 353, 356, 357, 359, 365, 368, 371, 372, 373, 375, 377, 379, 380, 382, 384,
		385 and 257141-265301.
33	Streptococcus pneumoniae	2, 10, 13, 25, 27, 33, 46, 48, 50, 52, 55, 62, 63, 67, 73, 81, 84, 91, 101, 105,
	TIGR4	106, 111, 119, 149, 152, 160, 161, 164, 166, 168, 169, 171, 172, 175, 176, 177,
		179, 180, 190, 205, 208, 213, 214, 218, 228, 236, 242, 244, 246, 262, 268, 271,
		272, 274, 297, 299, 306, 321, 323, 324, 325, 327, 329, 333, 340, 345, 348, 351,
		353, 356, 359, 365, 368, 371, 372, 375, 380 and 265302-266788.
34	Streptococcus pyogenes	3, 5, 8, 10, 21, 22, 25, 27, 32, 37, 38, 39, 40, 43, 49, 90, 95, 96, 106, 116,
	M1 GAS	126, 129, 138, 163, 164, 168, 175, 176, 180, 226, 232, 244, 246, 259, 261, 262,
		268, 283, 295, 296, 297, 299, 306, 309, 316, 321, 329, 330, 333, 348, 349, 359,
		372, 379, 380 and 266789-269521.
35	Streptococcus pyogenes	3, 8, 10, 13, 20, 22, 25, 27, 31, 32, 33, 37, 38, 40, 46, 48, 52, 55, 62, 67,
	MGAS315	73, 84, 90, 91, 105, 106, 113, 116, 129, 138, 152, 160, 164, 166, 168, 175, 176,
		177, 179, 180, 186, 190, 192, 205, 208, 211, 213, 214, 218, 226, 229, 232, 236,
		242, 244, 246, 262, 268, 271, 272, 274, 282, 283, 295, 296, 297, 299, 306, 309,
		312, 321, 323, 324, 325, 327, 329, 333, 340, 345, 348, 349, 353, 356, 359, 372,
		379, 380, 381 and 269522-272357.
36	Streptococcus pyogenes	3, 4, 8, 10, 13, 21, 22, 25, 27, 31, 33, 37, 38, 39, 40, 46, 48, 52, 55, 62, 67,
	MGAS8232	73, 84, 90, 91, 95, 105, 106, 113, 116, 129, 138, 152, 160, 163, 164, 166, 168,
		175, 176, 177, 179, 180, 190, 205, 208, 213, 214, 218, 226, 232, 236, 242, 244,
		246, 247, 259, 260, 261, 262, 268, 271, 272, 274, 295, 296, 297, 299, 306, 307,
		309, 316, 321, 323, 324, 325, 327, 329, 330, 333, 337, 340, 344, 345, 348, 349,
		353, 356, 359, 363, 372, 379, 380, 381 and 272358-275553.
37	Streptococcus pyogenes	10, 13, 25, 27, 31, 33, 46, 48, 52, 55, 62, 67, 73, 84, 91, 105, 113, 152, 160,
	SSI-1	164, 166, 168, 175, 176, 177, 179, 180, 190, 205, 208, 213, 214, 218, 236, 242,
		244, 246, 262, 268, 271, 272, 274, 297, 299, 306, 321, 323, 324, 325, 327, 329,
		333, 340, 345, 348, 353, 356, 359, 372, 380, 381 and 275554-276703.
38	Treponema pallidum subsp.	3, 10, 13, 48, 52, 57, 59, 67, 81, 84, 86, 90, 91, 121, 131, 134, 174, 175, 176,
	pallidum str.	184, 218, 228, 231, 235, 236, 243, 261, 262, 269, 272, 289, 291, 295, 299, 306,
	Nichols	312, 324, 329, 332, 333, 340, 345, 356, 358 and 276704-277654.
39	Yersinia pestis	1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 16, 18, 19, 21, 22, 25, 26, 27, 28, 29,
		30, 31, 32, 33, 34, 36, 37, 39, 40, 41, 42, 43, 45, 46, 47, 48, 51, 52, 53, 54,
		55, 57, 58, 61, 62, 63, 67, 68, 70, 71, 73, 75, 76, 78, 82, 84, 85, 87, 88, 89,
		90, 91, 93, 94, 95, 98, 99, 101, 102, 103, 105, 106, 107, 108, 111, 112, 113,
		114, 115, 116, 117, 120, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132, 133,
		134, 135, 136, 138, 140, 141, 142, 143, 146, 148, 149, 151, 152, 153, 154, 155,
		156, 160, 164, 165, 166, 167, 169, 171, 172, 174, 175, 176, 177, 178, 179, 180,
		182, 184, 186, 187, 188, 190, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202,
		203, 205, 206, 208, 209, 211, 213, 214, 215, 217, 218, 219, 220, 221, 222, 224,
		225, 226, 227, 229, 230, 232, 233, 234, 236, 237, 238, 239, 240, 241, 242, 243,
		244, 245, 250, 251, 252, 253, 255, 256, 257, 258, 259, 260, 262, 263, 264, 270,
		271, 272, 274, 276, 279, 280, 281, 282, 283, 286, 287, 289, 291, 292, 293, 295,
		296, 298, 299, 300, 301, 302, 304, 306, 307, 308, 309, 311, 314, 315, 317, 319,
		321, 322, 323, 324, 325, 326, 327, 329, 330, 331, 333, 334, 335, 336, 337, 340,
		341, 342, 343, 344, 345, 346, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,
		358, 359, 363, 364, 365, 367, 368, 370, 372, 373, 374, 376, 377, 378, 379, 380,
		381, 382, 383, 384 and 277655-287825.
40	Yersinia pestis KIM	1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 16, 18, 19, 20, 21, 22, 25, 26, 27, 28,
		29, 31, 32, 33, 34, 36, 37, 39, 40, 41, 42, 43, 45, 46, 47, 48, 51, 52, 53, 54,
		55, 57, 58, 61, 62, 63, 65, 67, 68, 70, 71, 72, 73, 75, 76, 78, 84, 85, 87, 88,
		89, 90, 91, 93, 94, 95, 97, 99, 101, 102, 103, 105, 106, 107, 108, 111, 112,
		113, 114, 115, 117, 118, 120, 121, 122, 123, 124, 125, 126, 129, 130, 131, 132,
		133, 134, 135, 136, 138, 140, 142, 143, 146, 147, 148, 149, 151, 152, 153, 154,
		156, 158, 160, 164, 165, 166, 169, 171, 172, 174, 175, 176, 177, 178, 179, 180,
		182, 186, 187, 188, 190, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202, 203,
		205, 206, 207, 208, 209, 211, 213, 214, 215, 217, 218, 220, 221, 222, 224, 225,
		226, 227, 229, 230, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,
		244, 247, 248, 250, 251, 252, 253, 255, 256, 257, 258, 260, 262, 263, 264, 270,
		271, 272, 274, 276, 279, 281, 282, 283, 284, 286, 287, 288, 289, 291, 292, 293,
		294, 295, 296, 298, 299, 300, 302, 303, 305, 306, 307, 308, 309, 311, 314, 315,
		317, 318, 319, 321, 322, 323, 324, 325, 327, 329, 330, 331, 333, 334, 335, 336,
		337, 340, 341, 342, 343, 344, 345, 346, 348, 349, 350, 351, 352, 353, 354, 355,
		356, 357, 358, 359, 362, 363, 364, 365, 367, 368, 370, 373, 374, 375, 376, 377,
		378, 379, 380, 381, 382, 383, 384, 385 and 287826-298021.

Claims

1. A bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.

2. A bioinformatically detectable isolated oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.

3. A bioinformatically detectable first oligonucleotide which is a portion of a mRNA transcript of a target gene, and anneals to a second oligonucleotide that is endogenously processed from a hairpin precursor, wherein binding of said first oligonucleotide to said second oligonucleotide represses expression of said target gene, and wherein nucleotide sequence of said second nucleotide is selected from the group consisting of SEQ ID NOs: 1-385 and 386-49787.

4. A bioinformatically detectable oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2337129-4223628.

5. A bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Bordetella pertussis infection, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 2.

6. A bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Brucella suis 1330 infection, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 3.

7. A bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydia trachomatis infection, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 4.

8. A bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae AR39 infection, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 5.

9. A bioinformatically detectable isolated oligonucleotide which anneals to a portion of a mRNA transcript of a target gene associated with Chlamydophila pneumoniae CWL029 infection, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide has at least 80% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs shown in Table 13 row 6.

10. A method for treatment of a disease involving a tissue in which a protein is pathologically expressed to an undesirable extent, said protein having a messenger RNA, the method comprising: providing a material which modulates activity of a microRNA oligonucleotide which binds complementarily to a segment of said messenger RNA; and introducing said material into said tissue, causing modulation of said activity of said microRNA oligonucleotide and thereby modulating expression of said protein in a desired manner.

11. A method for treatment of a disease involving tissue in which a protein is pathologically expressed to an undesirable extent, said protein having a messenger RNA, the method comprising: providing a material which at least partially binds a segment of said messenger RNA that is bound complementarily by a microRNA oligonucleotide, thereby modulating expression of said protein; and introducing said material into said tissue, thereby modulating expression of said protein.

12. A method for treatment of a disease involving a tissue in which a protein is pathologically over-expressed, said protein having a messenger RNA, the method comprising: providing a microRNA oligonucleotide which binds complementarily to a segment of said messenger RNA; and introducing said microRNA oligonucleotide into said tissue, causing said microRNA oligonucleotide to bind complementarily to a segment of said messenger RNA and thereby inhibit expression of said protein.

13. A method for treatment of a disease involving a tissue in which a protein is pathologically over-expressed, said protein having a messenger RNA, the method comprising: providing a chemically-modified microRNA oligonucleotide which binds complementarily to a segment of said messenger RNA; and introducing said chemically-modified microRNA oligonucleotide into said tissue, causing said microRNA oligonucleotide to bind complementarily to a segment of said messenger RNA and thereby inhibit expression of said protein.

14. A method for treatment of a disease involving a tissue in which a protein is pathologically under-expressed, said protein having a messenger RNA, the method comprising: providing an oligonucleotide that inhibits activity of a microRNA oligonucleotide which binds complementarily to a segment of said messenger RNA; and introducing said oligonucleotide into said tissue, causing inhibition of said activity of said microRNA oligonucleotide and thereby promotion of translation of said protein.

15. A method for treatment of a disease involving a tissue in which a protein is pathologically under-expressed, said protein having a messenger RNA, the method comprising: providing a chemically-modified oligonucleotide that inhibits activity of a microRNA oligonucleotide which binds complementarily to a segment of said messenger RNA; and introducing said chemically-modified oligonucleotide into said tissue, causing inhibition of said activity of said microRNA oligonucleotide and thereby promotion of translation of said protein.

16. A method for diagnosis of a disease involving a tissue in which a protein is expressed to abnormal extent, said protein having a messenger RNA, the method comprising: assaying a microRNA oligonucleotide which at least partially binds a segment of said messenger RNA and modulates expression of said protein, thereby providing an indication of at least one parameter of said disease.

17. A method for detection of expression of an oligonucleotide, the method comprising: determining a first nucleotide sequence of a first oligonucleotide, which first nucleotide sequence is not complementary to a genome of an organism; receiving a second nucleotide sequence of a second oligonucleotide whose expression is sought to be detected; designing a third nucleotide sequence that is complementary to said second nucleotide sequence of said second oligonucleotide, and a fourth nucleotide sequence that is complementary to a fifth nucleotide sequence which is different from said second nucleotide sequence of said second oligonucleotide by at least one nucleotide; synthesizing a first oligonucleotide probe having a sixth nucleotide sequence comprising said third nucleotide sequence followed by said first nucleotide sequence of said first oligonucleotide, and a second oligonucleotide probe having a seventh nucleotide sequence comprising said fourth nucleotide sequence followed by said first nucleotide sequence of said first oligonucleotide; locating said first oligonucleotide probe and said second oligonucleotide probe on a microarray platform; receiving an RNA test sample from at least one tissue of said organism; obtaining size-fractionated RNA from said RNA test sample; amplifying said size-fractionated RNA; hybridizing said adaptor-linked RNA with said first and second oligonucleotide probes on said microarray platform; and determining expression of said first oligonucleotide in said at least one tissue of said organism, based at least in part on said hybridizing.

18. A bioinformatically detectable isolated polynucleotide which is endogenously processed into a plurality of hairpin-shaped precursor oligonucleotides, each of which is endogenously processed into a respective oligonucleotide, which in turn anneals to a portion of a mRNA transcript of a target gene, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene.

19. A bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said target gene does not encode a protein.

20. A bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein a function of said oligonucleotide comprises modulation of cell type.

21. A bioinformatically detectable isolated oligonucleotide which is endogenously processed from a hairpin-shaped precursor, and anneals to a portion of a mRNA transcript of a target gene, wherein binding of said oligonucleotide to said mRNA transcript represses expression of said target gene, and wherein said oligonucleotide is maternally transferred by a cell to at least one daughter cell of said cell, and a function of said oligonucleotide comprises modulation of cell type of said daughter cell.

22. A method for bioinformatic detection of microRNA oligonucleotides, the method comprising: bioinformatically detecting a hairpin-shaped precursor oligonucleotide; bioinformatically detecting an oligonucleotide which is endogenously processed from said hairpin-shaped precursor oligonucleotide; and bioinformatically detecting a target gene of said oligonucleotide wherein said oligonucleotide anneals to at least one portion of a mRNA transcript of said target gene, and wherein said binding represses expression of said target gene, and said target gene is associated with a disease.